Exosome: A New Player in Translational Nanomedicine

Journal of Clinical Medicine Review Exosome: A New Player in Translational Nanomedicine Houssam Aheget 1,†, María Tristán-Manzano 1,†, Loubna Mazini 2, Marina Cortijo-Gutierrez 1, Pablo Galindo-Moreno 3, Concha Herrera 4,5, Francisco Martin 1, Juan Antonio Marchal 6,7,8,9 and Karim Benabdellah 1,* 1 Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Av. de la Illustration 114, 18016 Granada, Spain; [email protected] (H.A.); [email protected] (M.T.-M.); [email protected] (M.C.-G.); [email protected] (F.M.) 2Medical Application Interface Center, Mohammed VI Polytechnic University, 43152 Ben-Guerir, Morocco; [email protected] 3 Oral Surgery and Implant Dentistry Department, School of Dentistry, University of Granada, 18011 Granada, Spain; [email protected] 4Maimonides Institute of Biomedical Research in Cordoba (IMIBIC), 14004 Cordoba, Spain; [email protected] 5Department of Haematology, Reina Sofía University Hospital, 14004 Cordoba, Spain 6Biomedical Research Institute, ibs. Granada, 18012 Granada, Spain; [email protected] 7Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, 18016 Granada, Spain 8Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, 18016 Granada, Spain 9Excellence Research Unit Modeling Nature (MNat), University of Granada, 18016 Granada, Spain *Correspondence: [email protected]; Tel.: +34-958-715-500 †These authors contributed equally to this work. Received: 23 June 2020; Accepted: 22 July 2020; Published: 26 July 2020   Abstract: Summary: Exosomes are extracellular vesicles released by the vast majority of cell types both in vivo and ex vivo, upon the fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. Two main functions have been attributed to exosomes: their capacity to transport proteins, lipids and nucleic acids between cells and organs, as well as their potential to act as natural intercellular communicators in normal biological processes and in pathologies. From a clinical perspective, the majority of applications use exosomes as biomarkers of disease. A new approach uses exosomes as biologically active carriers to provide a platform for the enhanced delivery of cargo in vivo . One of the major limitations in developing exosome-based therapies is the difficulty of producing sufficient amounts of safe and efficient exosomes. The identification of potential proteins involved in exosome biogenesis is expected to directly cause a deliberate increase in exosome production. In this review, we summarize the current state of knowledge regarding exosomes, with particular emphasis on their structural features, biosynthesis pathways, production techniques and potential clinical applications. Keywords: immunotherapy; exosomes; CARs; gene editing; cancer; liquid biopsies 1. Introduction Extracellular vesicles (EVs) are differently sized vesicles released by the vast majority of cell types both in vivo and ex vivo. Two main functions have been attributed to EVs: (1) their capacity as natural intercellular communicators to transport proteins, lipids and nucleic acids between cells and organs in normal biological processes and (2) their active involvement in the progression of pathologies such as cancer. Based on their size, biogenesis pathways and other biophysical and biochemical criteria, EVs J. Clin. Med. 2020,9, 2380; doi:10.3390/jcm9082380 www.mdpi.com/journal/jcm J. Clin. Med. 2020,9, 2380 2 of 25 can be grouped into two main categories: microvesicles (MVs; 100–1000nm) and exosomes (EXOs; 30–100 nm) [1–3]. Microvesicles (MVs) can be distinguished from other EVs by their size and formation mechanisms, including cytoskeleton remodelling and phosphatidylserine externalization [ 4 , 5 ]. Like other EVs, MVs are derived from several cell types (Figure 1). Their formation is stimulated under specific conditions, by inflammatory processes and hypoxia among other stimuli [ 6 – 12 ], and they generally maintain the original cell-surface-specific antigens [ 13 – 19 ]. MVs play several physiological roles in the body through the transfer of active molecules, such as microRNA, proteins and lipids. These different functions enable MVs to regulate cellular processes including intercellular immune responses [ 20 , 21 ] angiogenesis [ 22 ], neuronal regeneration [ 23 ], anti-inflammatory protection [ 21 ] and coagulant mediation [ 24 ]. In addition to physiological processes, EVs are involved in intracellular degradation systems such as autophagy through specific signalling pathways [ 25 – 27 ] and the activation of molecules involved in apoptotic pathways [ 28 – 30 ]. Given the characteristics described above, MVs are clearly not just simple by-products of physiological and pathological processes, but are also key players in many different pathways. Here, we review the current state of knowledge concerning exosomes which are not directly shed from the parent cell plasma membrane, but rather are formed through a more complex process, with particular emphasis on their structural features, biosynthesis pathways, production techniques and potential clinical applications. J. Clin. Med. 2020, 9, 2380 2 of 27 as natural intercellular communicators to transport proteins, lipids and nucleic acids between cells and organs in normal biological processes and (2) their active involvement in the progression of pathologies such as cancer. Based on their size, biogenesis pathways and other biophysical and biochemical criteria, EVs can be grouped into two main categories: microvesicles (MVs; 100– 1000nm) and exosomes (EXOs; 30–100 nm) [1–3]. Microvesicles (MVs) can be distinguished from other EVs by their size and formation mechanisms, including cytoskeleton remodelling and phosphatidylserine externalization [4,5]. Like other EVs, MVs are derived from several cell types (Figure 1). Their formation is stimulated under specific conditions, by inflammatory processes and hypoxia among other stimuli [6–12], and they generally maintain the original cell-surface-specific antigens [13–19]. MVs play several physiological roles in the body through the transfer of active molecules, such as microRNA, proteins and lipids. These different functions enable MVs to regulate cellular processes including intercellular immune responses [20,21] angiogenesis [22], neuronal regeneration [23], antiinflammatory protection [21] and coagulant mediation [24]. In addition to physiological processes, EVs are involved in intracellular degradation systems such as autophagy through specific signalling pathways [25–27] and the activation of molecules involved in apoptotic pathways [28– 30]. Given the characteristics described above, MVs are clearly not just simple by-products of physiological and pathological processes, but are also key players in many different pathways. Here, we review the current state of knowledge concerning exosomes which are not directly shed from the parent cell plasma membrane, but rather are formed through a more complex process, with particular emphasis on their structural features, biosynthesis pathways, production techniques and potential clinical applications. Figure 1. Different exosome biogenesis pathways. Exosome formation begins with synteninsyndecan interactions which require direct interaction between ALIX and CHMP4 proteins. The intervention of two additional components, Tsg 101 (ESCRT-1) and Vps22 (ESCRT-II), has also been reported, although their mode of action remains little understood. Exosome formation is further regulated by heparanase, an enzyme that cleaves syndecan heparan sulfate, while the small GTase Figure 1. Different exosome biogenesis pathways. Exosome formation begins with syntenin-syndecan interactions which require direct interaction between ALIX and CHMP4 proteins. The intervention of two additional components, Tsg 101 (ESCRT-1) and Vps22 (ESCRT-II), has also been reported, although their mode of action remains little understood. Exosome formation is further regulated by heparanase, an enzyme that cleaves syndecan heparan sulfate, while the small GTase Arf6 also plays a crucial role. The small GTPase ADP ribosylation factor 6 (Arf6) and its effector phospholipase D2 (PLD2) regulate the syntenin pathway. The interaction of Arf6 and PLD2 affects exosome formation by controlling the J. Clin. Med. 2020,9, 2380 3 of 25 budding of intraluminal vesicles (ILVs) in multivesicular bodies (MVBs). The silencing of hepatocyte growth-factor-regulated tyrosine kinase substrate (Hrs)proteins, which interact with the tumour susceptibility gene 101 (tsg101) in exosome biogenesis, decreases the number of vesicles [ 31 ]. As interferon-stimulated gene 15 (Isg15) expression inhibits Tsg101 ubiquitination, the disruption of tsg15 may increase exosome release. The upregulation of the tumor-suppressor-activated pathway 6 (TSAP6), a p53-inducible transmembrane protein, has been shown to increase exosome production [ 32 ]. Two other possibilities are involved in ESCRT-independent pathway: the ceramide-based sphingomyelinase (SMase) pathway, in which sphingomyelin is hydrolysed into phosphorylcoline, and ceramide, which contributes to alternative exosome production. The third pathway is a tetraspanin-dependent pathway that involves CD63, belonging to the superfamily of tetraspanins, which, along with their partner molecules, form tetraspanin-enriched microdomains that contribute to exosome formation. Furthermore, exosome trafficking is regulated by the small GTPase, a member of the Rab and Ral protein superfamilies. For instance, Rab11, together with Rab27a/b, facilitate exovesicular secretion in a calcium-dependent manner [ 33 ]. Finally, SNARE and syntaxin 5 proteins enable vesicles to dock and fuse with the plasma membrane and to release exosomes into the external medium. 2. Exosome Biogenesis, Regulation and Function 2.1. Exosome Biogenesis Unlike MVs, exosomes constitute some of the most sophisticated intracellular trafficking systems (Figure 1). Exosome biogenesis takes place via plasma membrane (PM) invagination to form endosomes through the fusion of several primary vesicles. The maturation process occurs during the intracellular trafficking of endosomes from the PM to the centre of the cell, leading to overall changes in the lipid and protein composition of their cargo. In this regard, more than twenty proteins are involved and distributed through four Endosomal sorting complexes required for transport ESCRT (ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III), which complement the ESCRT-independent mechanism. Both pathways, which work synergistically, are involved in (1) protein sequestration and modification, (2) the processing and trafficking of the resulting vesicles, and (3) their fusion to the plasma membrane [ 33 ]. These mechanisms have been the subject of intense research in recent years. However, due to experimental challenges, it is unclear at which step the different molecules and enzymes are involved. Below, we discuss the different pathways involved in exosome biogenesis, sorting and release. We will also focus on proteins which may boost the production of exosomes or modulate their surface and content. Some non-ESCRT proteins, sphingolipids (SLs) and their metabolic enzymes will be highlighted as potential targets and as a new strategy to amplify exosome production. The components of the first protein complex, ESCRT-0, are the hepatocyte growth-factor-regulated tyrosine kinase substrate (Hrs), the signal-transducing adaptor molecule (STAM) and tumor susceptibility gene 101 (TSG101). This complex concentrates ubiquitylated cargo at specific micro-domains, thus facilitating the first step in membrane invagination [ 34 ]. This first phase involves a group of proteins, ALG-2-interacting protein X, (ALIX) syntenin and syndecan, which are involved in intraluminal vesicle (ILV) formation and cargo selection [ 35 ]. Depletion of ALIX proteins increases the amount of MHC-II in cells and, thus, in secreted vesicles, indicating that, rather than regulating vesicle biogenesis, ALIX proteins alter the exosomal membrane. This suggests that these proteins may be good candidates to modulate surface expression [ 36 ]. Other well-characterized proteins are syndecans (SDCs), type-I integral membrane heparan sulphate proteoglycans (HSPGs), composed of four genes (SDC 1-4). SDC 4 regulates several vesicular trafficking pathways together with syntenin and the adaptor protein Bro1/ALIX [ 37 ]. Transmembrane protein tumour-suppressor-activated pathway 6 (TSAP6), also identified as ferrireductase Steap3, plays a fundamental role during the first steps of the biogenesis pathway. This protein is strongly activated by DNA damage-activated transcription factor p53 in several cell lines [ 38 – 40 ]. TSAP6-deficient mice exhibit a phenotype associated with abnormal reticulocyte maturation, a process known to be dependent on exosome secretion [41]. J. Clin. Med. 2020,9, 2380 4 of 25 As mentioned above, several studies have highlighted the existence of at least two ESCRT-independent mechanisms in the initial steps of exosome formation involving lipids and tetraspanins. These protein superfamilies, together with a wide variety of transmembrane and cytosolic proteins, mediate the organization of tetraspanin-enriched microdomains (TEMS) in the plasma membrane and the biogenesis of exosomes [ 42 , 43 ]. Tetraspanins CD9, CD63, CD81, CD82 and CD151 are widely distributed among the different cell types, while others, such as Tssc6 CD37 and CD53, are restricted to specific tissues [ 44 ]. Exosome release by dendritic cells generated from CD9 knockout mice has been demonstrated to be lower than that from wild-type [ 45 ]. The knockdown of tetraspanin protein CD63 induces a significant increase in exosome production, thus confirming the important role played by CD63 in multivesicular endosome (MVE) biogenesis and/or trafficking [ 46 ]. The second ESCRT-independent mechanism involves ceramide, a simple sphingolipid (SL), which plays a critical role in membrane biogenesis [ 47 ]. SL biosynthesis starts in the endothelial reticulum (ER) with the condensation of L-serine and palmitoyl-CoA which are catalysed by serine palmitoyl-transferase (SPT), leading to the generation of a variety of long-chain SL bases. The resulting product is reduced by 3-ketosphinganine reductase and N-acylation, followed by a final reduction in dihydroceramide to ceramide mediated by dihydroceramide desaturase [ 48 ]. Ceramide can also be generated by sphingolipid hydrolysis through the intervention of sphingomyelinases (SMases) [49]. 2.2. Regulation and Function As mentioned above, as exosomes are highly sophisticated vesicles involved in numerous pathological and physiological processes, their secretion is strictly regulated and influenced by external and internal stimuli including biotic and abiotic stresses [ 50 ]. Trafficking events are governed by the Ras-associated binding (Rab) GTPase. Rab family proteins regulate the traffic pathways of different membrane compartments including exosomes. Rab27-deficient mice have been shown to be defective in several membrane-associated processes such as improper neutrophil chemotaxis. Rab27a-deficient natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) exhibit impaired cytotoxic granule exocytosis [ 51 , 52 ] and granule platelet release [ 53 , 54 ]. Five potential Rab GTPases, Rab2b, Rab5a, Rab9a, Rab27a and Rab27b, have been identified in HeLa cells during shRNA screening, targeting 59 GTPases, which play a major role in exosome secretion [ 55 ], with the involvement of Rab27a and Rab27b in exosome biogenesis attracting particular attention. A recent study reports that Rab27b, rather than Rab27a, regulates exosomal secretion in human umbilical vein endothelial cells (HUVECs) [ 56 ]. This and other studies have observed that Rab11 depletion severely diminishes exosome secretion in several cell types and have highlighted the role of Rab family proteins in exosome biogenesis. The Rho/Rac/cdc42 family of small membrane GTPases is also involved in the exosome pathway, with the RhoA effector citron kinase, in particular, observed to have a positive effect on exosome release [ 57 ]. The fusion of the resulting membrane with the plasma membrane (PM), as well as the release of exosomes to the extracellular medium, are both regulated by N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins [ 58 ]. Two SNARE protein family members, vesicular associated membrane protein 7 (VAMP7) and Synaptobrevin homolog YKT6 (YKT6), have been identified to play a major role in exosome release, specifically in human lung cancer cell lines, human embryonic kidney 293 cells (HEK293) and Adenocarcinomic human alveolar basal epithelial 549 cells (A549) [59,60]. External factors, such as the impact of viral infection on exosome production through mechanisms including the regulation of specific proteins, are closely associated with exosome biogenesis [ 61 – 63 ]. The hepatitis A virus (HAV) can hijack ALIX exosome-like pathways [ 64 , 65 ], while the respiratory syncytial virus (RSV) uses the exosome cargo to inhibit immune responses in the course of viral infection [ 66 ]. Bacterial and parasitic infections also affect exosome production and secretion [ 67 , 68 ], while metabolic dysfunction due to abiotic stress can also lead to exosomal changes. For example, environmental stresses, including ionizing radiation, can alter exosome secretion, composition and function [39,69–72]. J. Clin. Med. 2020,9, 2380 5 of 25 3. Different Types and Functions of Cells that Release Exosomes Exosomes can be produced by the vast majority of cells with different origins and numerous functions. The cells from which exosomes are secreted include T cells [ 73 , 74 ], platelets [ 75 ], megakaryocytes [ 76 ] mast cells [ 77 , 78 ], neurons [ 79 , 80 ], oligodendrocytes [ 81 ] and Schwann cells [ 82 – 85 ]. Similarly, cells with stemness properties, such as mesenchymal stromal cells (MSCs) [ 86 – 88 ] and induced pluripotent stem cells (iPSCs) [ 89 , 90 ], have been reported to release exosomes. In addition, exosomes are found in biological fluids including plasma [ 91 – 94 ], urine [ 95 – 97 ] saliva [ 98 ], amniotic fluid [ 99 ] and breast milk [ 100 ]. In the sections below, we provide details of major cells that play a key role in exosome secretion in different metabolic and pathological pathways. 3.1. Exosomes Derived from Mesenchymal Stem/Stromal Cells of Different Sources Involved in a Wide Range of Diseases and Metabolic Pathways Mesenchymal stromal cells (MSCs) are resident adult stem cells that have been identified in virtually all human tissues including bone marrow (BM), peripheral and cord blood (CB), dental pulp, liver and skin [ 101 ]. This explains their critical role in tissue repair and regeneration despite the differences observed in their population numbers, cell profiles and proliferation rates [102,103] . MSCs are characterized by self-renewal and differentiation capacity both in vitro and in vivo . However, their plasticity and heightened capacity to differentiate into endoderm and ectoderm cell layers have made MSCs of great interest for cell-based therapies in the field of regenerative medicine [ 104 – 106 ]. Although MSCs are known to be mediated through cell-cell communication, their secretome-rich cytokines, chemokines, micro-RNA, as well as different growth factors, involved in biological pathways, including cell proliferation, differentiation, migration and senescence [ 107 , 108 ], make them particularly suited to use in cell-based therapies. These secretomes are made up of extracellular vesicles (EVs) including exosomes [ 109 – 111 ], which can be characterized using specific International Society for Cellular Therapy (ISCT) guidelines. According to the ISCT, while positively expressing the stromal markers CD73 and CD105, MSCs negatively express the hematopoietic markers CD14, CD34 and CD45 [ 112 ]. MSCs are plastic-adherent cells of a fibroblast-like morphology capable of long-term expansion in culture and tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic progenitors. However, their immunogenicity is the most important reason for using MSCs in cell-based therapies. MSCs are immunosuppressive and inhibit T cell activation due to the lack of major histocompatibility complex (MHC) II [ 113 , 114 ]. Given the impaired expression of CD80 and CD86 in dendritic cells, in addition to B cell proliferation and differentiation, MSCs offer great potential for use in allogeneic transplantation [ 115 , 116 ]. Despite the usefulness of these criteria for identifying MSCs, these cell populations are reported to be heterogenous with regard to their non-clonal proliferation, differentiation potencies, stromal stem cell profiles and committed progenitors [ 117 , 118 ]. These differences appear to be related to their tissue origin and to the cell separation and culture expansion techniques used [ 119 , 120 ]. Thus, the complex composition of the exosomes released is markedly influenced by initial local cell crosstalk and microenvironmental priming. Despite clinical successes, consistency and safety issues remain a matter of debate [ 86 , 121 ]. MSC-derived exosomes present in conditioned media are considered an alternative to MSC-based therapies due to their superior efficiency and scalability [ 122 ]. More than 200 preclinical studies have been published as of July, 2020, on the applications of MSC-derived exosomes to a wide range of pathologies including neurological, cardiovascular, immunological and kidney diseases (https://clinicaltrials.gov/). MSC-derived exosomes obtained from different sources, particularly human bone-marrow (hBM) and human umbilical cord perivascular cells (hUCPVCs), have proven to have an impressive effect on neurological tissues, blood–brain barrier stability in lipopolysaccharide-induced neuroinflammation and on reactive astrogliosis [ 123 – 125 ]. Emerging evidence suggests that neurological disorders can be successfully treated by exosome-based therapy when the auto-regenerative capacity of the central nervous system (CNS) is limited [ 126 – 128 ]. BM-MSC-derived exosomes and human umbilical cord (hUC) MSC exosomes are also effectively used J. Clin. Med. 2020,9, 2380 6 of 25 for cardiac tissue neovascularization following ischemic injury [ 129 ] and for endothelial function enhancement, respectively [ 130 , 131 ]. Similarly, liver function is ameliorated by MSC-derived exosomes through the epithelial–mesenchymal transition (EMT) of hepatocyte and collagen production and through serum aspartate aminotransferase restoration [132–135]. 3.2. Exosomes Derived from Immune System-Related Cells That Play a Key Role in Several Immunological Processes Many immune cells, including T and B cells, macrophages, natural killers (NKs) and dendritic cells (DCs), are associated with exosome secretion capacity [ 136 ]. The existence of a set of proteins, such as CD63, Major histocompatibility complex class II (MHC-II), Fas ligand (FasL) and T cell receptor (TCR) (http://www.exocarta.org), on cellular surfaces provide further evidence of their role as mediators, modulators and activators in the immune system [ 137 , 138 ]. T cells, including CD4 + helper and CD8 + cytotoxic T cells, as well as regulatory T cells (Tregs), secrete exosomes and play different roles depending on T cell subtype origin and activation status (Figure 2). For example, exosomes derived from stimulated T cells can act as autologous signals to increase the proliferation of resting cells, resulting in an altered cytokine secretion profile [ 139 ]. Unlike classical T cells, exosomes derived from chimeric antigen receptor (CAR) T cells have recently been found to provide relatively safer therapies [ 140 , 141 ]. On the other hand, exosomes derived from the more antigen-specific subtype CD8 + T cell show antiviral activity associated with membrane proteins secreted via exosomes [ 142 ]. Moreover, CD4 + -associated exosomes have been reported to significantly boost B cell activation, proliferation and, thus, antibody production [ 143 , 144 ] and to act as immunoregulators [ 145 ]. Exosomes secreted by CD4 + CD25 + Foxp3 Treg cells, a subset of CD4 + T cells, specializing in immune tolerance establishment and maintenance, which use a diverse set of mechanisms to enforce peripheral tolerance, are thought to be deeply involved in immune regulation [ 146 ]. In the murine model, Tregs appear to produce quantitatively more exosomes than naïve CD4 + and CD8 + T cells [ 147 ]. Treg-derived exosomes also express the cell-surface enzyme CD73[ 148 , 149 ], which suppresses immune responses [ 150 ], the IL-2 receptor chain CD25, which plays a key role in autoimmune disease suppression [ 151 ], and T Lymphocyte-associated antigen-4 (CTLA-4), an immune inhibitory factor constitutively expressed in Tregs [152]. Both lymphoid and myeloid lineages have the capacity to secrete exosomes. Several studies have highlighted the potential of B cells to secrete exosomes carrying the peptide pMHC-II, in addition to costimulatory and adhesion molecules. This type of exosome induces antigen-specific MHC class II-restricted T cell responses [ 153 ]. The second potential target of B-cell-derived exosomes is dendritic cells (DCs) which present MHC-II peptides to CD4 + T cells, T cell-derived exosome-DC reciprocal interactions begin with DC priming by exosomes which contain genomic and mitochondrial DNA through antigen-driven contacts [ 154 ]. Exosomes derived from a specific subset of T cells may regulate other T cell subtypes. In addition, T-cell-derived exosomes specifically inhibit viral transcription through the presence of antiviral membrane-bound factors [ 142 ]. Mast cells constitutively release exosomes expressing CD63 and OX40 ligand (OX40L), which promote CD4 + T cell proliferation, thus facilitating T helper 2 (Th2) cell differentiation [ 155 ]. DCs are also able to secrete exosomes which express, on their surface, MHC-peptide complexes, T cells costimulatory molecules and other compounds which interact with immune cells. Furthermore, macrophages secrete exosomes with proinflammatory activity when secreted by M1 macrophages (M1) and with anti-inflammatory activity when secreted by M2 macrophages (M2). Natural killer (NK) cells, members of the lymphoid cell family, play a major role in innate immunity and tumor progression control through their cytolytic activity, cytokine production and by improving T-helper 1 responses. Exosomes produced from NK cells and their apoptotic activity in tumor cells have also been studied. Exosomes obtained from IL-2and IL-15-stimulated NK cells were detected in peripheral blood expressing the typical NK-related molecules CD16, CD69 and NKG2D which have high penetrance rates and a marked cytolytic effect on tumor sites [ 156 ]. NK-derived exosomes J. Clin. Med. 2020,9, 2380 7 of 25 carrying the tumor suppressor microRNA (miR)-186 exhibit potent activity against neuroblastoma cell lines [ 157 ] and aggressive melanoma in vitro and in vivo [ 158 ], thus opening up the possibility of clinical applications using the antitumor activity of NK-derived exosomes [ 159 ]. Dendritic cells (DCs), another powerful cancer immunotherapy tool, have recently become an alternative source of exosomes [ 160 , 161 ]. In fact, several groups have demonstrated the feasibility of obtaining exosomes from DCs [ 162 ] to alleviate the clinical symptoms of various diseases [ 163 – 168 ]. These exosomes alleviate the effects of hepatic ischemia/reperfusion (I/R) injury by modulating Treg/Th17 cell balance [ 169 ] and also induce transplantation immune tolerance [170–172]. J. Clin. Med. 2020, 9, 2380 7 of 27 Figure 2. Secretion of exosomes associated with immune cells types and their modes of action. Both lymphoid and myeloid lineages have the capacity to secrete exosomes. Several studies have highlighted the potential of B cells to secrete exosomes carrying the peptide pMHC-II, in addition to costimulatory and adhesion molecules. This type of exosome induces antigen-specific MHC class II-restricted T cell responses [153]. The second potential target of B-cell-derived exosomes is dendritic cells (DCs) which present MHC-II peptides to CD4 + T cells, T cell-derived exosome-DC reciprocal interactions begin with DC priming by exosomes which contain genomic and mitochondrial DNA through antigen-driven contacts [154]. Exosomes derived from a specific subset of T cells may regulate other T cell subtypes. In addition, T-cell-derived exosomes specifically inhibit viral transcription through the presence of antiviral membrane-bound factors [142]. Mast cells constitutively release exosomes expressing CD63 and OX40 ligand (OX40L), which promote CD4 + T cell proliferation, thus facilitating T helper 2 (Th2) cell differentiation [155]. DCs are also able to secrete exosomes which express, on their surface, MHC-peptide complexes, T cells Figure 2. Secretion of exosomes associated with immune cells types and their modes of action. Finally, macrophages, a diverse cell population found in specific organs and the blood stream, can be divided into two sub-groups: classically activated macrophages (M1) and alternatively activated macrophages (M2), induced by type 1 T helper cells (Th1) and by type 2 T helper cells (Th2), respectively. Despite their proinflammatory profile, M1 macrophages polarize to M2 status given their anti-inflammatory capacity to respond to microenvironmental stimuli during inflammation-associated diseases. M1 macrophages secrete elevated levels of proinflammatory cytokines such as tumor necrosis J. Clin. Med. 2020,9, 2380 8 of 25 factor-alpha (TNF-), interleukin (IL)-1 and IL-6; M2 macrophages, composed of four subgroups M2a, M2b, M2c and M2d, secrete immunoregulatory cytokines such as IL-10 [ 173 ]. Exosomes recently obtained from both types of these multi-functional macrophages [ 174 ], isolated from M2a and M2b, presented strong anti-inflammatory activity, through the Th2 activation and immunoregulation [ 175 ]. In addition, M2a-exosomes are capable of regulating the behaviour of breast cancer cells by inducing or reversing their dormancy [176]. 4. Exosome Manufacturing Status and Challenges Based on their role in intercellular communication, especially with stem cells in their microenvironment, exosomes are expected to play a critical role in the regulation of numerous physiological and pathological processes. Their use in new disease therapy strategies presents many challenges, mainly with regard to regulatory production guidelines, qualified staffand the marketing strategies required for this widely used type of therapy. Another challenge is the difficulty of classifying exosome-based therapies which differ enormously from cell-based treatments. In terms of size, exosomes are likely to be in the form of platelet lysates, ranging from whole cells to pharmaceutical molecules. Quality control is likely to combine current Good Manufacturing Practice (cGMP) guidelines for cells and traditional cGMP for pharmaceutical drugs [ 177 , 178 ]. European regulatory agencies and the Food and Drug Administration (FDA), classify human use extracellular vesicles (EVs) as biological medications, have laid down a regulatory framework for manufacture and clinical trials in the journal of the International Society for Extracellular Vesicles (ISEV) published recently [ 179 ]. In their 2018 guidelines, ISEV states that the following information is required for each EV formulation: (i) precise data concerning origin, including the number of secreting cells, biofluid volume and tissue mass, (ii) precise EVs abundance data, including total particle numbers and/or protein and lipid content, (iii) presence of components associated with EV subtypes and EVs generically, depending on the specific intended function and iv) the presence of non-vesicular and co-isolated components [180]. The heterogenous composition of exosomes, containing mostly proteins and nucleic acids, is subject to potency and quality testing, similar to that used for current cellular therapies [ 178 ]. It is of critical importance to define the pharmaceutical classification of active substances responsible for the effects of the therapy in order to determine the pharmaceutical quality control strategy to be used for exosome production [ 181 , 182 ]. These active substances can be overexpressed by genome editing to improve homogeneity, purity and manufacturing reproducibility. The homogeneity assays should also be optimized to track the biomarkers selected in each batch and to identify non-active ingredients. The general production conditions for these products, which are similar to those for drug manufacturing, include current GMP guidelines, large-scale production characterized by high reproducibility, scalability, stability, storage, banking and clinical quality control in allogenic settings, as reported elsewhere [ 177 , 183 ]. The collection, separation, expansion storage and point-of-care transfer of stem cell progenitors and mature cells need to be integrated into therapeutic strategies and strictly controlled and regulated to ensure patient safety and to maintain the sustainable therapeutic efficacy of the purified exosomes. 4.1. Large Scale Production of Exosomes One of the major challenges in developing exosome-based therapies is the need to produce a sufficient number of safe and efficient exosomes. Depending on the disease and condition of the patient, considerable quantities of exosomes will be required to provide adequate treatment. For example, EVs were administered to a patient with graft-versus-host disease (GVHD) in progressively increasing doses, beginning with a total protein dose of 0.05–0.15 mg/kg and ending with a dose of 0.20–0.60 mg/kg [ 184 ]. The appropriate dose of exosome proteins is dependent upon the disease of the patient and associated factors. Exosomes are also extracted from the culture media of large-scale biotechnological waste products [ 185 – 187 ]. To boost the production of cell culture exosomes, the expanded stem, progenitor and mature cells need to be adequately activated by growth factors, J. Clin. Med. 2020,9, 2380 9 of 25 nutrients, oxygen concentrations and other stimuli. The long-term expansion of MSCs affects their morphology, stem cell-associated profile, proliferation and clonogenic capacity [ 188 ], as well as the links between cell profile and physiological changes. Some evidence indicates that early expansion of MSCs is related to their stemness profile, while long-term expanded MSCs are stromal cells associated with a senescent phenotype [ 188 , 189 ]. Recent studies report increasing proliferation induced by younger MSC-derived secretome, probably due to the secretion of rejuvenating growth factors such as GDF11 [ 190 , 191 ]. Interestingly, other studies have suggested that the secretome of senescent MSCs has a biological effect on their microenvironment [ 103 , 189 , 192 , 193 ]. This senescence messaging secretome, or senescence-associated secretory phenotype (SASP), probably induces changes in the cellular transcriptional program [ 194 ] which, in turn, lead to changes in the number and composition of EVs, thus reflecting the senescent profile of the parent MSCs [ 192 , 193 , 195 ]. SASPs have also been reported to play an effective role in inducing senescence in immortalized prostate cells [ 193 ]. Alessio et al. (2019) have also identified an increase in IGFBP-4 protein levels in the SASP caused by genotoxic stress and initial senescence status [ 196 ]. Thus, to better adapt exosomes to their intended therapeutic function, SASPs should be avoided during culture production. Nevertheless, MSC culture expansion is critical for pooling the appropriate number of cells, mainly in closed-culture bioreactors, suggesting that the avoidance of SASPs during exosome production has become an important issue. The use of mesenchymal hematopoietic and endothelial markers to sort cells could be useful for purifying specific MSC populations. However, as with bone marrow (BM)-MSCs, initial MSC identification of the sorted subpopulations could limit yields and availability. Gene editing could therefore be essential to increase the number of exosomes produced per MSC with reduced cell culture throughput, thus avoiding SASP development during long-term culture expansion. Another technical hurdle is to be surmounted is the need to reduce artifacts when using differential ultracentrifugation [ 197 ], gel-filtration on special matrices [ 198 ] and size-exclusion chromatography (SEC) for exosome separation and concentration. These techniques have recently been reported to completely transform the basic composition of exosomes [ 199 ]. However, tangential flow filtration (TFF) and SEC purification, which appear to be best adapted to large-scale production [ 200 ], are used commercially to produce recombinant proteins and antibodies, methods which might also be suitable for exosome purification. For enhanced specificity, additional techniques are combined with primary steps such as washing and ultrafiltration [ 201 – 203 ]. High-resolution density gradient fractionation and direct immunoaffinity capture can also be used for EV analysis and characterization [204]. 4.2. High-Quality Uniform Exosomes MSC-derived exosome therapies are largely dependent upon the regenerative and immunomodulatory capacities of MSCs. Some evidence shows that hypoxia, nutrient starvation and microenvironment changes in pH enhance the release of EVs such as exosomes [ 204 ]. Exosomes can also be engineered to take advantage of their natural production processes and properties combined with genetic and non-genetic techniques to add new functionalities. Different active procedures are used to selectively enrich exosome cargo with miRNAs or small-molecule drugs. A poly (A)-binding protein is used to selectively recruit mRNAs into exosomes, while a zipcode-like 25 nucleotide (nt) sequence can be incorporated into the three prime untranslated region (3 0 UTR) of the mRNA of interest and be recruited by Z-DNA binding protein 1 (ZBP1) [ 205 ]. Receptor–ligand pairs are also used to deliver modified exosomes presenting membrane-bound ligand receptors to target surface cells [ 206 , 207 ]. Exosomes can also be enhanced by active loading through electroporation [ 208 ] and chemical conjugation [ 209 ]. These molecular biology techniques raise the expression of the protein and nucleic acid of interest well above physiological expression levels. However, the production of uniform exosomes with regard to their composition, membrane markers and even size, is difficult but not impossible. Further research is critical to improve molecular targeting in different aspects of exosome biogenesis and its involvement in biological processes. Interestingly, exosomes can also be J. Clin. Med. 2020,9, 2380 16 of 25 38. Jella, K.K.; Rani, S.; O’Driscoll, L.; McClean, B.; Byrne, H.J.; Lyng, F.M. Exosomes are involved in mediating radiation induced bystander signaling in human keratinocyte cells. Radiat. Res. 2014 ,181, 138–145. [CrossRef] 39. Lehmann, B.D.; Paine, M.S.; Brooks, A.M.; McCubrey, J.A.; Renegar, R.H.; Wang, R.; Terrian, D.M. Senescence-associated exosome release from human prostate cancer cells. Cancer Res. 2008 ,68, 7864–7871. [CrossRef] 40. Al-Mayah, A.; Bright, S.; Chapman, K.; Irons, S.; Luo, P.; Carter, D.; Goodwin, E.; Kadhim, M. The non-targeted effects of radiation are perpetuated by exosomes. Mutat. Res. 2015,772, 38–45. [CrossRef] 41. Lespagnol, A.; Duflaut, D.; Beekman, C.; Blanc, L.; Fiucci, G.; Marine, J.C.; Vidal, M.; Amson, R.; Telerman, A. Exosome secretion, including the DNA damage-induced p53-dependent secretory pathway, is severely compromised in TSAP6/Steap3-null mice. Cell Death Differ. 2008,15, 1723–1733. [CrossRef] 42. Charrin, S.; le Naour, F.; Silvie, O.; Milhiet, P.E.; Boucheix, C.; Rubinstein, E. Lateral organization of membrane proteins: Tetraspanins spin their web. Biochem. J. 2009,420, 133–154. [CrossRef] [PubMed] 43. Hemler, M.E. Tetraspanin functions and associated microdomains. Nat. Rev. Mol. Cell Biol. 2005 ,6, 801–811. [CrossRef] [PubMed] 44. Schaper, F.; van Spriel, A.B. Antitumor Immunity Is Controlled by Tetraspanin Proteins. Front. Immunol. 2018,9, 1185. [CrossRef] [PubMed] 45. Chairoungdua, A.; Smith, D.L.; Pochard, P.; Hull, M.; Caplan, M.J. Exosome release of beta-catenin: A novel mechanism that antagonizes Wnt signaling. J. Cell Biol. 2010,190, 1079–1091. [CrossRef] [PubMed] 46. Petersen, S.H.; Odintsova, E.; Haigh, T.A.; Rickinson, A.B.; Taylor, G.S.; Berditchevski, F. The role of tetraspanin CD63 in antigen presentation via MHC class II. Eur. J. Immunol. 2011 ,41, 2556–2561. [CrossRef] 47. Trajkovic, K.; Hsu, C.; Chiantia, S.; Rajendran, L.; Wenzel, D.; Wieland, F.; Schwille, P.; Brugger, B.; Simons, M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 2008 ,319, 1244–1247. [CrossRef] 48. Loewith, R.; Riezman, H.; Winssinger, N. Sphingolipids and membrane targets for therapeutics. Curr. Opin. Chem. Biol. 2019,50, 19–28. [CrossRef] 49. Clarke, C.J. Neutral Sphingomyelinases in Cancer: Friend or Foe? Adv. Cancer Res. 2018 ,140, 97–119. [CrossRef] 50. Gatta, A.T.; Carlton, J.G. The ESCRT-machinery: Closing holes and expanding roles. Curr. Opin. Cell Biol. 2019,59, 121–132. [CrossRef] 51. Singh, R.K.; Furze, R.C.; Birrell, M.A.; Rankin, S.M.; Hume, A.N.; Seabra, M.C. A role for Rab27 in neutrophil chemotaxis and lung recruitment. BMC Cell Biol. 2014,15, 39. [CrossRef] [PubMed] 52. Alexander, M.; Ramstead, A.G.; Bauer, K.M.; Lee, S.H.; Runtsch, M.C.; Wallace, J.; Huffaker, T.B.; Larsen, D.K.; Tolmachova, T.; Seabra, M.C.; et al. Rab27-Dependent Exosome Production Inhibits Chronic Inflammation and Enables Acute Responses to Inflammatory Stimuli. J. Immunol. 2017 ,199, 3559–3570. [CrossRef] [PubMed] 53. Adam, F.; Kauskot, A.; Kurowska, M.; Goudin, N.; Munoz, I.; Bordet, J.C.; Huang, J.D.; Bryckaert, M.; Fischer, A.; Borgel, D.; et al. Kinesin-1 Is a New Actor Involved in Platelet Secretion and Thrombus Stability. Arterioscler. Thromb. Vasc. Biol. 2018,38, 1037–1051. [CrossRef] [PubMed] 54. Al Hawas, R.; Ren, Q.; Ye, S.; Karim, Z.A.; Filipovich, A.H.; Whiteheart, S.W. Munc18b/STXBP2 is required for platelet secretion. Blood 2012,120, 2493–2500. [CrossRef] [PubMed] 55. Ostrowski, M.; Carmo, N.B.; Krumeich, S.; Fanget, I.; Raposo, G.; Savina, A.; Moita, C.F.; Schauer, K.; Hume, A.N.; Freitas, R.P.; et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 2010,12, 19–30. [CrossRef] [PubMed] 56. Jae, N.; McEwan, D.G.; Manavski, Y.; Boon, R.A.; Dimmeler, S. Rab7a and Rab27b control secretion of endothelial microRNA through extracellular vesicles. FEBS Lett. 2015,589, 3182–3188. [CrossRef] 57. Loomis, R.J.; Holmes, D.A.; Elms, A.; Solski, P.A.; Der, C.J.; Su, L. Citron kinase, a RhoA effector, enhances HIV-1 virion production by modulating exocytosis. Traffic2006,7, 1643–1653. [CrossRef] 58. Baker, R.W.; Hughson, F.M. Chaperoning SNARE assembly and disassembly. Nat. Rev. Mol. Cell Biol. 2016 , 17, 465–479. [CrossRef] 59. Fader, C.M.; Sanchez, D.G.; Mestre, M.B.; Colombo, M.I. TI-VAMP/VAMP7 and VAMP3/cellubrevin: Two v-SNARE proteins involved in specific steps of the autophagy/multivesicular body pathways. Biochim. Biophys. Acta 2009,1793, 1901–1916. [CrossRef] 60. Gross, J.C.; Chaudhary, V.; Bartscherer, K.; Boutros, M. Active Wnt proteins are secreted on exosomes. Nat. Cell Biol. 2012,14, 1036–1045. [CrossRef] J. Clin. Med. 2020,9, 2380 17 of 25 61. Alenquer, M.; Amorim, M.J. Exosome Biogenesis, Regulation, and Function in Viral Infection. Viruses 2015 ,7, 5066–5083. [CrossRef] 62. Ghossoub, R.; Lembo, F.; Rubio, A.; Gaillard, C.B.; Bouchet, J.; Vitale, N.; Slavik, J.; Machala, M.; Zimmermann, P. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2. Nat. Commun. 2014,5, 3477. [CrossRef] [PubMed] 63. Glingston, R.S.; Deb, R.; Kumar, S.; Nagotu, S. Organelle dynamics and viral infections: At cross roads. Microbes. Infect. 2019,21, 20–32. [CrossRef] [PubMed] 64. Jiang, W.; Ma, P.; Deng, L.; Liu, Z.; Wang, X.; Liu, X.; Long, G. Hepatitis A virus structural protein pX interacts with ALIX and promotes the secretion of virions and foreign proteins through exosome-like vesicles. J. Extracell. Vesicles 2020,9, 1716513. [CrossRef] [PubMed] 65. Feng, Z.; Hensley, L.; McKnight, K.L.; Hu, F.; Madden, V.; Ping, L.; Jeong, S.H.; Walker, C.; Lanford, R.E.; Lemon, S.M. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 2013 , 496, 367–371. [CrossRef] [PubMed] 66. Chahar, H.S.; Corsello, T.; Kudlicki, A.S.; Komaravelli, N.; Casola, A. Respiratory Syncytial Virus Infection Changes Cargo Composition of Exosome Released from Airway Epithelial Cells. Sci. Rep. 2018 ,8, 387. [CrossRef] 67. Babatunde, K.A.; Yesodha Subramanian, B.; Ahouidi, A.D.; Martinez Murillo, P.; Walch, M.; Mantel, P.Y. Role of Extracellular Vesicles in Cellular Cross Talk in Malaria. Front. Immunol. 2020,11, 22. [CrossRef] 68. Jiang, N.; Yu, S.; Yang, N.; Feng, Y.; Sang, X.; Wang, Y.; Wahlgren, M.; Chen, Q. Characterization of the Catalytic Subunits of the RNA Exosome-like Complex in Plasmodium falciparum. J. Eukaryot. Microbiol. 2018,65, 843–853. [CrossRef] 69. Ariyoshi, K.; Miura, T.; Kasai, K.; Fujishima, Y.; Nakata, A.; Yoshida, M. Radiation-Induced Bystander Effect is Mediated by Mitochondrial DNA in Exosome-Like Vesicles. Sci. Rep. 2019,9, 9103. [CrossRef] 70. Mo, L.J.; Song, M.; Huang, Q.H.; Guan, H.; Liu, X.D.; Xie, D.F.; Huang, B.; Huang, R.X.; Zhou, P.K. Exosome-packaged miR-1246 contributes to bystander DNA damage by targeting LIG4. Br. J. Cancer 2018 , 119, 492–502. [CrossRef] 71. Xu, S.; Wang, J.; Ding, N.; Hu, W.; Zhang, X.; Wang, B.; Hua, J.; Wei, W.; Zhu, Q. Exosome-mediated microRNA transfer plays a role in radiation-induced bystander effect. RNA Biol. 2015 ,12, 1355–1363. [CrossRef] [PubMed] 72. Goulielmaki, E.; Ioannidou, A.; Tsekrekou, M.; Stratigi, K.; Poutakidou, I.K.; Gkirtzimanaki, K.; Aivaliotis, M.; Evangelou, K.; Topalis, P.; Altmuller, J.; et al. Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage. Nat. Commun. 2020,11, 42. [CrossRef] [PubMed] 73. Chen, L.; Huang, H.; Zhang, W.; Ding, F.; Fan, Z.; Zeng, Z. Exosomes Derived From T Regulatory Cells Suppress CD8+Cytotoxic T Lymphocyte Proliferation and Prolong Liver Allograft Survival. Med. Sci. Monit. 2019,25, 4877–4884. [CrossRef] [PubMed] 74. Xie, Y.; Zhang, X.; Zhao, T.; Li, W.; Xiang, J. Natural CD8(+)25(+) regulatory T cell-secreted exosomes capable of suppressing cytotoxic T lymphocyte-mediated immunity against B16 melanoma. Biochem. Biophys. Res. Commun. 2013,438, 152–155. [CrossRef] 75. Zhang, J.P.; Chen, B.B.; Zheng, H.H.; Yang, H.J.; Song, L. [Discharge and Contents of Exosome from Leukocyte-Reduced Apheresis Platelet Stored for Different Storage Time]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2019,27, 1993–1997. [CrossRef] 76. Kovuru, N.; Raghuwanshi, S.; Gutti, R.K. Exosome mediated differentiation of megakaryocytes: A study on TLR mediated effects. J. Thromb. Thrombolysis 2019,48, 171–173. [CrossRef] 77. Shelke, G.V.; Yin, Y.; Jang, S.C.; Lasser, C.; Wennmalm, S.; Hoffmann, H.J.; Li, L.; Gho, Y.S.; Nilsson, J.A.; Lotvall, J. Endosomal signalling via exosome surface TGFbeta-1. J. Extracell. Vesicles 2019,8, 1650458. [CrossRef] 78. Chen, B.; Li, M.Y.; Guo, Y.; Zhao, X.; Liu, Y.Y. Mast cell-derived exosome participates in acupoint-stimulation initiated local network activities. Zhen Ci Yan Jiu 2015,40, 82–85. 79. Mathews, P.M.; Levy, E. Exosome Production Is Key to Neuronal Endosomal Pathway Integrity in Neurodegenerative Diseases. Front. Neurosci. 2019,13, 1347. [CrossRef] 80. Bowers, E.C.; Hassanin, A.A.I.; Ramos, K.S. In vitro models of exosome biology and toxicology: New frontiers in biomedical research. Toxicol. In Vitro 2020,64, 104462. [CrossRef] 81. Benjamins, J.A.; Nedelkoska, L.; Touil, H.; Stemmer, P.M.; Carruthers, N.J.; Jena, B.P.; Naik, A.R.; Bar-Or, A.; Lisak, R.P. Exosome-enriched fractions from MS B cells induce oligodendrocyte death. Neurol. Neuroimmunol. Neuroinflamm. 2019,6, e550. [CrossRef] [PubMed] J. Clin. Med. 2020,9, 2380 18 of 25 82. Sohn, E.J.; Park, H.T.; Shin, Y.K. Exosomes derived from differentiated Schwann cells inhibit Schwann cell migration via microRNAs. Neuroreport 2020,31, 515–522. [CrossRef] [PubMed] 83. Wang, L.; Chopp, M.; Szalad, A.; Lu, X.; Zhang, Y.; Wang, X.; Cepparulo, P.; Lu, M.; Li, C.; Zhang, Z.G. Exosomes Derived From Schwann Cells Ameliorate Peripheral Neuropathy in Type 2 Diabetic Mice. Diabetes 2020,69, 749–759. [CrossRef] [PubMed] 84. Zhou, M.; Hu, M.; He, S.; Li, B.; Liu, C.; Min, J.; Hong, L. Effects of RSC96 Schwann Cell-Derived Exosomes on Proliferation, Senescence, and Apoptosis of Dorsal Root Ganglion Cells In Vitro. Med. Sci. Monit. 2018 , 24, 7841–7849. [CrossRef] 85. Jia, L.; Chopp, M.; Wang, L.; Lu, X.; Szalad, A.; Zhang, Z.G. Exosomes derived from high-glucose-stimulated Schwann cells promote development of diabetic peripheral neuropathy. FASEB J. 2018 ,32, 6911–6922. [CrossRef] 86. Elahi, F.M.; Farwell, D.G.; Nolta, J.A.; Anderson, J.D. Preclinical translation of exosomes derived from mesenchymal stem/stromal cells. Stem Cells 2020,38, 15–21. [CrossRef] 87. Vilaca-Faria, H.; Salgado, A.J.; Teixeira, F.G. Mesenchymal Stem Cells-derived Exosomes: A New Possible Therapeutic Strategy for Parkinson’s Disease? Cells 2019,8, 118. [CrossRef] 88. Yu, B.; Zhang, X.; Li, X. Exosomes derived from mesenchymal stem cells. Int. J. Mol. Sci. 2014 ,15, 4142–4157. [CrossRef] 89. Yang, P.C. Induced Pluripotent Stem Cell (iPSC)-Derived Exosomes for Precision Medicine in Heart Failure. Circ. Res. 2018,122, 661–663. [CrossRef] 90. Jung, J.H.; Fu, X.; Yang, P.C. Exosomes Generated From iPSC-Derivatives: New Direction for Stem Cell Therapy in Human Heart Diseases. Circ. Res. 2017,120, 407–417. [CrossRef] 91. Lin, X.; Ling, Q.; Lv, Y.; Ye, W.; Huang, J.; Li, X.; Guo, Q.; Wang, J.; Li, Z.; Jin, J. Plasma exosome-derived microRNA-532 as a novel predictor for acute myeloid leukemia. Cancer Biomark. 2020 ,28, 1–8. [CrossRef] [PubMed] 92. Bebelman, M.P.; Bun, P.; Huveneers, S.; van Niel, G.; Pegtel, D.M.; Verweij, F.J. Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells. Nat. Protoc. 2020,15, 102–121. [CrossRef] [PubMed] 93. Asare-Werehene, M.; Nakka, K.; Reunov, A.; Chiu, C.T.; Lee, W.T.; Abedini, M.R.; Wang, P.W.; Shieh, D.B.; Dilworth, F.J.; Carmona, E.; et al. The exosome-mediated autocrine and paracrine actions of plasma gelsolin in ovarian cancer chemoresistance. Oncogene 2020,39, 1600–1616. [CrossRef] 94. Garcia-Contreras, M.; Shah, S.H.; Tamayo, A.; Robbins, P.D.; Golberg, R.B.; Mendez, A.J.; Ricordi, C. Plasma-derived exosome characterization reveals a distinct microRNA signature in long duration Type 1 diabetes. Sci. Rep. 2017,7, 5998. [CrossRef] 95. Yazarlou, F.; Mowla, S.J.; Oskooei, V.K.; Motevaseli, E.; Tooli, L.F.; Afsharpad, M.; Nekoohesh, L.; Sanikhani, N.S.; Ghafouri-Fard, S.; Modarressi, M.H. Urine exosome gene expression of cancer-testis antigens for prediction of bladder carcinoma. Cancer Manag. Res. 2018 ,10, 5373–5381. [CrossRef] [PubMed] 96. Street, J.M.; Koritzinsky, E.H.; Glispie, D.M.; Yuen, P.S.T. Urine Exosome Isolation and Characterization. Methods Mol. Biol. 2017,1641, 413–423. [CrossRef] [PubMed] 97. McKiernan, J.; Donovan, M.J.; O’Neill, V.; Bentink, S.; Noerholm, M.; Belzer, S.; Skog, J.; Kattan, M.W.; Partin, A.; Andriole, G.; et al. A Novel Urine Exosome Gene Expression Assay to Predict High-grade Prostate Cancer at Initial Biopsy. JAMA Oncol. 2016,2, 882–889. [CrossRef] 98. Yang, J.; Wei, F.; Schafer, C.; Wong, D.T. Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva. PLoS ONE 2014,9, e110641. [CrossRef] 99. Dixon, C.L.; Sheller-Miller, S.; Saade, G.R.; Fortunato, S.J.; Lai, A.; Palma, C.; Guanzon, D.; Salomon, C.; Menon, R. Amniotic Fluid Exosome Proteomic Profile Exhibits Unique Pathways of Term and Preterm Labor. Endocrinology 2018,159, 2229–2240. [CrossRef] 100. Torregrosa Paredes, P.; Gutzeit, C.; Johansson, S.; Admyre, C.; Stenius, F.; Alm, J.; Scheynius, A.; Gabrielsson, S. Differences in exosome populations in human breast milk in relation to allergic sensitization and lifestyle. Allergy 2014,69, 463–471. [CrossRef] 101. Mastrolia, I.; Foppiani, E.M.; Murgia, A.; Candini, O.; Samarelli, A.V.; Grisendi, G.; Veronesi, E.; Horwitz, E.M.; Dominici, M. Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review. Stem Cells Transl. Med. 2019,8, 1135–1148. [CrossRef] [PubMed] J. Clin. Med. 2020,9, 2380 19 of 25 102. de Araujo Farias, V.; Carrillo-Galvez, A.B.; Martin, F.; Anderson, P. TGF-beta and mesenchymal stromal cells in regenerative medicine, autoimmunity and cancer. Cytokine Growth Factor Rev. 2018 ,43, 25–37. [CrossRef] [PubMed] 103. Squillaro, T.; Peluso, G.; Galderisi, U. Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transplant. 2016,25, 829–848. [CrossRef] [PubMed] 104. D’Souza, N.; Rossignoli, F.; Golinelli, G.; Grisendi, G.; Spano, C.; Candini, O.; Osturu, S.; Catani, F.; Paolucci, P.; Horwitz, E.M.; et al. Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies. BMC Med. 2015,13, 186. [CrossRef] [PubMed] 105. Vishnubalaji, R.; Al-Nbaheen, M.; Kadalmani, B.; Aldahmash, A.; Ramesh, T. Comparative investigation of the differentiation capability of bone-marrowand adipose-derived mesenchymal stem cells by qualitative and quantitative analysis. Cell Tissue Res. 2012,347, 419–427. [CrossRef] 106. Vishnubalaji, R.; Manikandan, M.; Al-Nbaheen, M.; Kadalmani, B.; Aldahmash, A.; Alajez, N.M. In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells. BMC Dev. Biol. 2012,12, 7. [CrossRef] 107. Amable, P.R.; Teixeira, M.V.; Carias, R.B.; Granjeiro, J.M.; Borojevic, R. Mesenchymal stromal cell proliferation, gene expression and protein production in human platelet-rich plasma-supplemented media. PLoS ONE 2014,9, e104662. [CrossRef] 108. Amable, P.R.; Teixeira, M.V.; Carias, R.B.; Granjeiro, J.M.; Borojevic, R. Protein synthesis and secretion in human mesenchymal cells derived from bone marrow, adipose tissue and Wharton’s jelly. Stem Cell Res. Ther. 2014,5, 53. [CrossRef] [PubMed] 109. Barreca, M.M.; Cancemi, P.; Geraci, F. Mesenchymal and Induced Pluripotent Stem Cells-Derived Extracellular Vesicles: The New Frontier for Regenerative Medicine? Cells 2020,9, 1163. [CrossRef] [PubMed] 110. Cai, J.; Wu, J.; Wang, J.; Li, Y.; Hu, X.; Luo, S.; Xiang, D. Extracellular vesicles derived from different sources of mesenchymal stem cells: Therapeutic effects and translational potential. Cell Biosci. 2020 ,10, 69. [CrossRef] [PubMed] 111. Lelek, J.; Zuba-Surma, E.K. Perspectives for Future Use of Extracellular Vesicles from Umbilical Cordand Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells in Regenerative Therapies-Synthetic Review. Int. J. Mol. Sci. 2020,21, 799. [CrossRef] [PubMed] 112. Dominici, M.; Le Blanc, K.; Mueller, I.; Slaper-Cortenbach, I.; Marini, F.; Krause, D.; Deans, R.; Keating, A.; Prockop, D.; Horwitz, E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006,8, 315–317. [CrossRef] [PubMed] 113. Jacobs, S.A.; Roobrouck, V.D.; Verfaillie, C.M.; Van Gool, S.W. Immunological characteristics of human mesenchymal stem cells and multipotent adult progenitor cells. Immunol. Cell Biol. 2013 ,91, 32–39. [CrossRef] [PubMed] 114. Jiang, X.X.; Zhang, Y.; Liu, B.; Zhang, S.X.; Wu, Y.; Yu, X.D.; Mao, N. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005,105, 4120–4126. [CrossRef] 115. Spaggiari, G.M.; Abdelrazik, H.; Becchetti, F.; Moretta, L. MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: Central role of MSC-derived prostaglandin E2. Blood 2009,113, 6576–6583. [CrossRef] 116. Bartholomew, A.; Sturgeon, C.; Siatskas, M.; Ferrer, K.; McIntosh, K.; Patil, S.; Hardy, W.; Devine, S.; Ucker, D.; Deans, R.; et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp. Hematol. 2002,30, 42–48. [CrossRef] 117. Alessio, N.; Pipino, C.; Mandatori, D.; Di Tomo, P.; Ferone, A.; Marchiso, M.; Melone, M.A.B.; Peluso, G.; Pandolfi, A.; Galderisi, U. Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study. J. Cell. Physiol. 2018,233, 8996–9006. [CrossRef] 118. Squillaro, T.; Antonucci, I.; Alessio, N.; Esposito, A.; Cipollaro, M.; Melone, M.A.B.; Peluso, G.; Stuppia, L.; Galderisi, U. Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes. J. Cell. Physiol. 2017,232, 3454–3467. [CrossRef] 119. Khong, D.; Li, M.; Singleton, A.; Chin, L.Y.; Mukundan, S.; Parekkadan, B. Orthogonal potency analysis of mesenchymal stromal cell function during ex vivo expansion. Exp. Cell Res. 2018,362, 102–110. [CrossRef] J. Clin. Med. 2020,9, 2380 20 of 25 120. Pachon-Pena, G.; Yu, G.; Tucker, A.; Wu, X.; Vendrell, J.; Bunnell, B.A.; Gimble, J.M. Stromal stem cells from adipose tissue and bone marrow of age-matched female donors display distinct immunophenotypic profiles. J. Cell. Physiol. 2011,226, 843–851. [CrossRef] 121. Neri, S. Genetic Stability of Mesenchymal Stromal Cells for Regenerative Medicine Applications: A Fundamental Biosafety Aspect. Int. J. Mol. Sci. 2019,20, 2406. [CrossRef] [PubMed] 122. Casado-Diaz, A.; Quesada-Gomez, J.M.; Dorado, G. Extracellular Vesicles Derived From Mesenchymal Stem Cells (MSC) in Regenerative Medicine: Applications in Skin Wound Healing. Front. Bioeng. Biotechnol. 2020 , 8, 146. [CrossRef] [PubMed] 123. Wang, H.; Zheng, R.; Chen, Q.; Shao, J.; Yu, J.; Hu, S. Mesenchymal stem cells microvesicles stabilize endothelial barrier function partly mediated by hepatocyte growth factor (HGF). Stem Cell Res. Ther. 2017 ,8, 211. [CrossRef] [PubMed] 124. Park, H.J.; Shin, J.Y.; Kim, H.N.; Oh, S.H.; Song, S.K.; Lee, P.H. Mesenchymal stem cells stabilize the blood-brain barrier through regulation of astrocytes. Stem Cell Res. Ther. 2015,6, 187. [CrossRef] 125. Xian, P.; Hei, Y.; Wang, R.; Wang, T.; Yang, J.; Li, J.; Di, Z.; Liu, Z.; Baskys, A.; Liu, W.; et al. Mesenchymal stem cell-derived exosomes as a nanotherapeutic agent for amelioration of inflammation-induced astrocyte alterations in mice. Theranostics 2019,9, 5956–5975. [CrossRef] 126. Ribeiro, C.A.; Fraga, J.S.; Graos, M.; Neves, N.M.; Reis, R.L.; Gimble, J.M.; Sousa, N.; Salgado, A.J. The secretome of stem cells isolated from the adipose tissue and Wharton jelly acts differently on central nervous system derived cell populations. Stem Cell Res. Ther. 2012,3, 18. [CrossRef] 127. Huang, J.H.; Yin, X.M.; Xu, Y.; Xu, C.C.; Lin, X.; Ye, F.B.; Cao, Y.; Lin, F.Y. Systemic Administration of Exosomes Released from Mesenchymal Stromal Cells Attenuates Apoptosis, Inflammation, and Promotes Angiogenesis after Spinal Cord Injury in Rats. J. Neurotrauma. 2017,34, 3388–3396. [CrossRef] 128. Teixeira, F.G.; Carvalho, M.M.; Sousa, N.; Salgado, A.J. Mesenchymal stem cells secretome: A new paradigm for central nervous system regeneration? Cell. Mol. Life Sci. 2013,70, 3871–3882. [CrossRef] 129. Teng, X.; Chen, L.; Chen, W.; Yang, J.; Yang, Z.; Shen, Z. Mesenchymal Stem Cell-Derived Exosomes Improve the Microenvironment of Infarcted Myocardium Contributing to Angiogenesis and Anti-Inflammation. Cell. Physiol. Biochem. 2015,37, 2415–2424. [CrossRef] 130. Qu, Q.; Pang, Y.; Zhang, C.; Liu, L.; Bi, Y. Exosomes derived from human umbilical cord mesenchymal stem cells inhibit vein graft intimal hyperplasia and accelerate reendothelialization by enhancing endothelial function. Stem Cell Res. Ther. 2020,11, 133. [CrossRef] 131. Xiong, Z.H.; Wei, J.; Lu, M.Q.; Jin, M.Y.; Geng, H.L. Protective effect of human umbilical cord mesenchymal stem cell exosomes on preserving the morphology and angiogenesis of placenta in rats with preeclampsia. Biomed. Pharmacother. 2018,105, 1240–1247. [CrossRef] [PubMed] 132. Shao, M.; Xu, Q.; Wu, Z.; Chen, Y.; Shu, Y.; Cao, X.; Chen, M.; Zhang, B.; Zhou, Y.; Yao, R.; et al. Exosomes derived from human umbilical cord mesenchymal stem cells ameliorate IL-6-induced acute liver injury through miR-455-3p. Stem Cell Res. Ther. 2020,11, 37. [CrossRef] [PubMed] 133. Zhang, L.; Song, Y.; Chen, L.; Li, D.; Feng, H.; Lu, Z.; Fan, T.; Chen, Z.; Livingston, M.J.; Geng, Q. MiR-20a-containing exosomes from umbilical cord mesenchymal stem cells alleviates liver ischemia/reperfusion injury. J. Cell. Physiol. 2020,235, 3698–3710. [CrossRef] [PubMed] 134. Jiang, L.; Zhang, S.; Hu, H.; Yang, J.; Wang, X.; Ma, Y.; Jiang, J.; Wang, J.; Zhong, L.; Chen, M.; et al. Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acute liver failure by reducing the activity of the NLRP3 inflammasome in macrophages. Biochem. Biophys. Res. Commun. 2019 ,508, 735–741. [CrossRef] [PubMed] 135. Li, T.; Yan, Y.; Wang, B.; Qian, H.; Zhang, X.; Shen, L.; Wang, M.; Zhou, Y.; Zhu, W.; Li, W.; et al. Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis. Stem Cells Dev. 2013 ,22, 845–854. [CrossRef] [PubMed] 136. Thery, C.; Ostrowski, M.; Segura, E. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 2009,9, 581–593. [CrossRef] 137. Lu, J.; Wu, J.; Tian, J.; Wang, S. Role of T cell-derived exosomes in immunoregulation. Immunol. Res. 2018 ,66, 313–322. [CrossRef] 138. Ventimiglia, L.N.; Alonso, M.A. Biogenesis and Function of T Cell-Derived Exosomes. Front. Cell Dev. Biol. 2016,4, 84. [CrossRef] J. Clin. Med. 2020,9, 2380 21 of 25 139. Wahlgren, J.; Karlson Tde, L.; Glader, P.; Telemo, E.; Valadi, H. Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling. PLoS ONE 2012,7, e49723. [CrossRef] 140. Fu, W.; Lei, C.; Liu, S.; Cui, Y.; Wang, C.; Qian, K.; Li, T.; Shen, Y.; Fan, X.; Lin, F.; et al. CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity. Nat. Commun. 2019,10, 4355. [CrossRef] 141. Tang, X.J.; Sun, X.Y.; Huang, K.M.; Zhang, L.; Yang, Z.S.; Zou, D.D.; Wang, B.; Warnock, G.L.; Dai, L.J.; Luo, J. Therapeutic potential of CAR-T cell-derived exosomes: A cell-free modality for targeted cancer therapy. Oncotarget 2015,6, 44179–44190. [CrossRef] [PubMed] 142. Tumne, A.; Prasad, V.S.; Chen, Y.; Stolz, D.B.; Saha, K.; Ratner, D.M.; Ding, M.; Watkins, S.C.; Gupta, P. Noncytotoxic suppression of human immunodeficiency virus type 1 transcription by exosomes secreted from CD8+T cells. J. Virol. 2009,83, 4354–4364. [CrossRef] [PubMed] 143. Lu, J.; Wu, J.; Xie, F.; Tian, J.; Tang, X.; Guo, H.; Ma, J.; Xu, P.; Mao, L.; Xu, H.; et al. CD4(+) T Cell-Released Extracellular Vesicles Potentiate the Efficacy of the HBsAg Vaccine by Enhancing B Cell Responses. Adv. Sci. (Weinh.) 2019,6, 1802219. [CrossRef] [PubMed] 144. Li, R.; Chibbar, R.; Xiang, J. Novel EXO-T vaccine using polyclonal CD4(+) T cells armed with HER2-specific exosomes for HER2-positive breast cancer. Onco Targ. Ther. 2018,11, 7089–7093. [CrossRef] [PubMed] 145. Zhang, H.; Xie, Y.; Li, W.; Chibbar, R.; Xiong, S.; Xiang, J. CD4(+) T cell-released exosomes inhibit CD8(+) cytotoxic T-lymphocyte responses and antitumor immunity. Cell. Mol. Immunol. 2011,8, 23–30. [CrossRef] 146. Li, P.; Liu, C.; Yu, Z.; Wu, M. New Insights into Regulatory T Cells: Exosomeand Non-Coding RNA-Mediated Regulation of Homeostasis and Resident Treg Cells. Front. Immunol. 2016,7, 574. [CrossRef] 147. Okoye, I.S.; Coomes, S.M.; Pelly, V.S.; Czieso, S.; Papayannopoulos, V.; Tolmachova, T.; Seabra, M.C.; Wilson, M.S. MicroRNA-containing T-regulatory-cell-derived exosomes suppress pathogenic T helper 1 cells. Immunity 2014,41, 89–103. [CrossRef] 148. Smyth, L.A.; Ratnasothy, K.; Tsang, J.Y.; Boardman, D.; Warley, A.; Lechler, R.; Lombardi, G. CD73 expression on extracellular vesicles derived from CD4+CD25+Foxp3+T cells contributes to their regulatory function. Eur. J. Immunol. 2013,43, 2430–2440. [CrossRef] 149. Clayton, A.; Al-Taei, S.; Webber, J.; Mason, M.D.; Tabi, Z. Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production. J. Immunol. 2011,187, 676–683. [CrossRef] 150. Stagg, J.; Divisekera, U.; Duret, H.; Sparwasser, T.; Teng, M.W.; Darcy, P.K.; Smyth, M.J. CD73-deficient mice have increased antitumor immunity and are resistant to experimental metastasis. Cancer Res. 2011 ,71, 2892–2900. [CrossRef] 151. Yu, A.; Snowhite, I.; Vendrame, F.; Rosenzwajg, M.; Klatzmann, D.; Pugliese, A.; Malek, T.R. Selective IL-2 responsiveness of regulatory T cells through multiple intrinsic mechanisms supports the use of low-dose IL-2 therapy in type 1 diabetes. Diabetes 2015,64, 2172–2183. [CrossRef] [PubMed] 152. Wing, K.; Onishi, Y.; Prieto-Martin, P.; Yamaguchi, T.; Miyara, M.; Fehervari, Z.; Nomura, T.; Sakaguchi, S. CTLA-4 control over Foxp3+regulatory T cell function. Science 2008,322, 271–275. [CrossRef] 153. Raposo, G.; Nijman, H.W.; Stoorvogel, W.; Liejendekker, R.; Harding, C.V.; Melief, C.J.; Geuze, H.J. B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 1996,183, 1161–1172. [CrossRef] [PubMed] 154. Torralba, D.; Baixauli, F.; Villarroya-Beltri, C.; Fernandez-Delgado, I.; Latorre-Pellicer, A.; Acin-Perez, R.; Martin-Cofreces, N.B.; Jaso-Tamame, A.L.; Iborra, S.; Jorge, I.; et al. Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts. Nat. Commun. 2018,9, 2658. [CrossRef] [PubMed] 155. Li, F.; Wang, Y.; Lin, L.; Wang, J.; Xiao, H.; Li, J.; Peng, X.; Dai, H.; Li, L. Mast Cell-Derived Exosomes Promote Th2 Cell Differentiation via OX40L-OX40 Ligation. J. Immunol. Res. 2016,2016, 3623898. [CrossRef] 156. Pace, A.L.D.; Tumino, N.; Besi, F.; Alicata, C.; Conti, L.A.; Munari, E.; Maggi, E.; Vacca, P.; Moretta, L. Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor In Exosome-Mediated Cytotoxicity Against Tumor. Cancers (Basel) 2020,12, 661. [CrossRef] 157. Neviani, P.; Wise, P.M.; Murtadha, M.; Liu, C.W.; Wu, C.H.; Jong, A.Y.; Seeger, R.C.; Fabbri, M. Natural Killer-Derived Exosomal miR-186 Inhibits Neuroblastoma Growth and Immune Escape Mechanisms. Cancer Res. 2019,79, 1151–1164. [CrossRef] 158. Zhu, L.; Kalimuthu, S.; Gangadaran, P.; Oh, J.M.; Lee, H.W.; Baek, S.H.; Jeong, S.Y.; Lee, S.W.; Lee, J.; Ahn, B.C. Exosomes Derived From Natural Killer Cells Exert Therapeutic Effect in Melanoma. Theranostics 2017 ,7, 2732–2745. [CrossRef] J. Clin. Med. 2020,9, 2380 22 of 25 159. Tian, H.; Li, W. Dendritic cell-derived exosomes for cancer immunotherapy: Hope and challenges. Ann. Transl. Med. 2017,5, 221. [CrossRef] 160. Pitt, J.M.; Charrier, M.; Viaud, S.; Andre, F.; Besse, B.; Chaput, N.; Zitvogel, L. Dendritic cell-derived exosomes as immunotherapies in the fight against cancer. J. Immunol. 2014,193, 1006–1011. [CrossRef] 161. Pitt, J.M.; Andre, F.; Amigorena, S.; Soria, J.C.; Eggermont, A.; Kroemer, G.; Zitvogel, L. Dendritic cell-derived exosomes for cancer therapy. J. Clin. Investig. 2016,126, 1224–1232. [CrossRef] [PubMed] 162. Leone, D.A.; Rees, A.J.; Kain, R. Dendritic cells and routing cargo into exosomes. Immunol. Cell Biol. 2018 ,96, 683–693. [CrossRef] [PubMed] 163. Li, X.L.; Li, H.; Zhang, M.; Xu, H.; Yue, L.T.; Zhang, X.X.; Wang, S.; Wang, C.C.; Li, Y.B.; Dou, Y.C.; et al. Exosomes derived from atorvastatin-modified bone marrow dendritic cells ameliorate experimental autoimmune myasthenia gravis by up-regulated levels of IDO/Treg and partly dependent on FasL/Fas pathway. J. Neuroinflamm. 2016,13, 8. [CrossRef] [PubMed] 164. Wang, L.; Yu, Z.; Wan, S.; Wu, F.; Chen, W.; Zhang, B.; Lin, D.; Liu, J.; Xie, H.; Sun, X.; et al. Exosomes Derived from Dendritic Cells Treated with Schistosoma japonicum Soluble Egg Antigen Attenuate DSS-Induced Colitis. Front. Pharmacol. 2017,8, 651. [CrossRef] 165. Li, J.; Huang, S.; Zhou, Z.; Lin, W.; Chen, S.; Chen, M.; Ye, Y. Exosomes derived from rAAV/AFP-transfected dendritic cells elicit specific T cell-mediated immune responses against hepatocellular carcinoma. Cancer Manag. Res. 2018,10, 4945–4957. [CrossRef] 166. Liu, H.; Chen, L.; Peng, Y.; Yu, S.; Liu, J.; Wu, L.; Zhang, L.; Wu, Q.; Chang, X.; Yu, X.; et al. Dendritic cells loaded with tumor derived exosomes for cancer immunotherapy. Oncotarget 2018 ,9, 2887–2894. [CrossRef] 167. Li, H.; Luo, Y.; Zhu, L.; Hua, W.; Zhang, Y.; Zhang, H.; Zhang, L.; Li, Z.; Xing, P.; Hong, B.; et al. Glia-derived exosomes: Promising therapeutic targets. Life Sci. 2019,239, 116951. [CrossRef] 168. Shi, S.; Rao, Q.; Zhang, C.; Zhang, X.; Qin, Y.; Niu, Z. Dendritic Cells Pulsed with Exosomes in Combination with PD-1 Antibody Increase the Efficacy of Sorafenib in Hepatocellular Carcinoma Model. Transl. Oncol. 2018,11, 250–258. [CrossRef] 169. Zheng, L.; Li, Z.; Ling, W.; Zhu, D.; Feng, Z.; Kong, L. Exosomes Derived from Dendritic Cells Attenuate Liver Injury by Modulating the Balance of Treg and Th17 Cells After Ischemia Reperfusion. Cell. Physiol. Biochem. 2018,46, 740–756. [CrossRef] 170. Pang, X.L.; Wang, Z.G.; Liu, L.; Feng, Y.H.; Wang, J.X.; Xie, H.C.; Yang, X.L.; Li, J.F.; Feng, G.W. Immature dendritic cells derived exosomes promotes immune tolerance by regulating T cell differentiation in renal transplantation. Aging (Albany N. Y.) 2019,11, 8911–8924. [CrossRef] 171. Agarwal, A.; Fanelli, G.; Letizia, M.; Tung, S.L.; Boardman, D.; Lechler, R.; Lombardi, G.; Smyth, L.A. Regulatory T cell-derived exosomes: Possible therapeutic and diagnostic tools in transplantation. Front. Immunol. 2014 ,5, 555. [CrossRef] [PubMed] 172. Yang, X.; Meng, S.; Jiang, H.; Zhu, C.; Wu, W. Exosomes derived from immature bone marrow dendritic cells induce tolerogenicity of intestinal transplantation in rats. J. Surg. Res. 2011 ,171, 826–832. [CrossRef] [PubMed] 173. Wynn, T.A.; Chawla, A.; Pollard, J.W. Macrophage biology in development, homeostasis and disease. Nature 2013,496, 445–455. [CrossRef] [PubMed] 174. Kim, H.; Wang, S.Y.; Kwak, G.; Yang, Y.; Kwon, I.C.; Kim, S.H. Exosome-Guided Phenotypic Switch of M1 to M2 Macrophages for Cutaneous Wound Healing. Adv. Sci. (Weinh.) 2019,6, 1900513. [CrossRef] 175. Yang, R.; Liao, Y.; Wang, L.; He, P.; Hu, Y.; Yuan, D.; Wu, Z.; Sun, X. Exosomes Derived From M2b Macrophages Attenuate DSS-Induced Colitis. Front. Immunol. 2019,10, 2346. [CrossRef] 176. Walker, N.D.; Elias, M.; Guiro, K.; Bhatia, R.; Greco, S.J.; Bryan, M.; Gergues, M.; Sandiford, O.A.; Ponzio, N.M.; Leibovich, S.J.; et al. Exosomes from differentially activated macrophages influence dormancy or resurgence of breast cancer cells within bone marrow stroma. Cell Death Dis. 2019,10, 59. [CrossRef] 177. Aiastui, A. Should Cell Culture Platforms Move towards EV Therapy Requirements? Front. Immunol. 2015 , 6, 8. [CrossRef] 178. Sahin, U.; Kariko, K.; Tureci, O. mRNA-based therapeutics-developing a new class of drugs. Nat. Rev. Drug Discov. 2014,13, 759–780. [CrossRef] 179. Lener, T.; Gimona, M.; Aigner, L.; Borger, V.; Buzas, E.; Camussi, G.; Chaput, N.; Chatterjee, D.; Court, F.A.; Del Portillo, H.A.; et al. Applying extracellular vesicles based therapeutics in clinical trials—An ISEV position paper. J. Extracell. Vesicles 2015,4, 30087. [CrossRef] J. Clin. Med. 2020,9, 2380 23 of 25 180. Thery, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018,7, 1535750. [CrossRef] 181. Jiang, X.R.; Song, A.; Bergelson, S.; Arroll, T.; Parekh, B.; May, K.; Chung, S.; Strouse, R.; Mire-Sluis, A.; Schenerman, M. Advances in the assessment and control of the effector functions of therapeutic antibodies. Nat. Rev. Drug Discov. 2011,10, 101–111. [CrossRef] [PubMed] 182. Gardner, C.R.; Walsh, C.T.; Almarsson, O. Drugs as materials: Valuing physical form in drug discovery. Nat. Rev. Drug Discov. 2004,3, 926–934. [CrossRef] [PubMed] 183. Katsuda, T.; Kosaka, N.; Takeshita, F.; Ochiya, T. The therapeutic potential of mesenchymal stem cell-derived extracellular vesicles. Proteomics 2013,13, 1637–1653. [CrossRef] [PubMed] 184. Kordelas, L.; Rebmann, V.; Ludwig, A.K.; Radtke, S.; Ruesing, J.; Doeppner, T.R.; Epple, M.; Horn, P.A.; Beelen, D.W.; Giebel, B. MSC-derived exosomes: A novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 2014,28, 970–973. [CrossRef] [PubMed] 185. Smith, Z.J.; Lee, C.; Rojalin, T.; Carney, R.P.; Hazari, S.; Knudson, A.; Lam, K.; Saari, H.; Ibanez, E.L.; Viitala, T.; et al. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content. J. Extracell. Vesicles 2015,4, 28533. [CrossRef] 186. Lobb, R.J.; Becker, M.; Wen, S.W.; Wong, C.S.; Wiegmans, A.P.; Leimgruber, A.; Moller, A. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles 2015 ,4, 27031. [CrossRef] 187. Jong, A.Y.; Wu, C.H.; Li, J.; Sun, J.; Fabbri, M.; Wayne, A.S.; Seeger, R.C. Large-scale isolation and cytotoxicity of extracellular vesicles derived from activated human natural killer cells. J. Extracell. Vesicles 2017 ,6, 1294368. [CrossRef] 188. Bogdanova, A.; Berzins, U.; Nikulshin, S.; Skrastina, D.; Ezerta, A.; Legzdina, D.; Kozlovska, T. Characterization of human adipose-derived stem cells cultured in autologous serum after subsequent passaging and long term cryopreservation. J. Stem Cells 2014,9, 135–148. 189. Lunyak, V.V.; Amaro-Ortiz, A.; Gaur, M. Mesenchymal Stem Cells Secretory Responses: Senescence Messaging Secretome and Immunomodulation Perspective. Front. Genet. 2017,8, 220. [CrossRef] 190. Rochette, L.; Mazini, L.; Meloux, A.; Zeller, M.; Cottin, Y.; Vergely, C.; Malka, G. Anti-Aging Effects of GDF11 on Skin. Int. J. Mol. Sci. 2020,21, 2598. [CrossRef] 191. Zarei, F.; Abbaszadeh, A. Application of Cell Therapy for Anti-Aging Facial Skin. Curr. Stem Cell Res. Ther. 2019,14, 244–248. [CrossRef] [PubMed] 192. Boulestreau, J.; Maumus, M.; Rozier, P.; Jorgensen, C.; Noel, D. Mesenchymal Stem Cell Derived Extracellular Vesicles in Aging. Front. Cell Dev. Biol. 2020,8, 107. [CrossRef] [PubMed] 193. Alessio, N.; Aprile, D.; Squillaro, T.; Di Bernardo, G.; Finicelli, M.; Melone, M.A.; Peluso, G.; Galderisi, U. The senescence-associated secretory phenotype (SASP) from mesenchymal stromal cells impairs growth of immortalized prostate cells but has no effect on metastatic prostatic cancer cells. Aging (Albany N. Y.) 2019 , 11, 5817–5828. [CrossRef] [PubMed] 194. Acosta, J.C.; Snijders, A.P.; Gil, J. Unbiased characterization of the senescence-associated secretome using SILAC-based quantitative proteomics. Methods Mol. Biol. 2013,965, 175–184. [CrossRef] 195. Lei, Q.; Liu, T.; Gao, F.; Xie, H.; Sun, L.; Zhao, A.; Ren, W.; Guo, H.; Zhang, L.; Wang, H.; et al. Microvesicles as Potential Biomarkers for the Identification of Senescence in Human Mesenchymal Stem Cells. Theranostics 2017,7, 2673–2689. [CrossRef] 196. Alessio, N.; Squillaro, T.; Di Bernardo, G.; Galano, G.; De Rosa, R.; Melone, M.A.; Peluso, G.; Galderisi, U. Increase of circulating IGFBP-4 following genotoxic stress and its implication for senescence. Elife 2020 ,9, e54523. [CrossRef] 197. Cvjetkovic, A.; Lotvall, J.; Lasser, C. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J. Extracell. Vesicles 2014,3, 23111. [CrossRef] 198. Nordin, J.Z.; Lee, Y.; Vader, P.; Mager, I.; Johansson, H.J.; Heusermann, W.; Wiklander, O.P.; Hallbrink, M.; Seow, Y.; Bultema, J.J.; et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 2015 ,11, 879–883. [CrossRef] 199. Jeppesen, D.K.; Fenix, A.M.; Franklin, J.L.; Higginbotham, J.N.; Zhang, Q.; Zimmerman, L.J.; Liebler, D.C.; Ping, J.; Liu, Q.; Evans, R.; et al. Reassessment of Exosome Composition. Cell 2019 ,177, 428–445. [CrossRef] J. Clin. Med. 2020,9, 2380 24 of 25 200. Heinemann, M.L.; Ilmer, M.; Silva, L.P.; Hawke, D.H.; Recio, A.; Vorontsova, M.A.; Alt, E.; Vykoukal, J. Benchtop isolation and characterization of functional exosomes by sequential filtration. J. Chromatogr. A 2014,1371, 125–135. [CrossRef] 201. Morales-Kastresana, A.; Telford, B.; Musich, T.A.; McKinnon, K.; Clayborne, C.; Braig, Z.; Rosner, A.; Demberg, T.; Watson, D.C.; Karpova, T.S.; et al. Labeling Extracellular Vesicles for Nanoscale Flow Cytometry. Sci. Rep. 2017,7, 1878. [CrossRef] 202. Corso, G.; Mager, I.; Lee, Y.; Gorgens, A.; Bultema, J.; Giebel, B.; Wood, M.J.A.; Nordin, J.Z.; Andaloussi, S.E. Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography. Sci. Rep. 2017,7, 11561. [CrossRef] [PubMed] 203. Vergauwen, G.; Dhondt, B.; Van Deun, J.; De Smedt, E.; Berx, G.; Timmerman, E.; Gevaert, K.; Miinalainen, I.; Cocquyt, V.; Braems, G.; et al. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Sci. Rep. 2017,7, 2704. [CrossRef] [PubMed] 204. Mills, J.; Capece, M.; Cocucci, E.; Tessari, A.; Palmieri, D. Cancer-Derived Extracellular Vesicle-Associated MicroRNAs in Intercellular Communication: One Cell’s Trash Is Another Cell’s Treasure. Int. J. Mol. Sci. 2019,20, 6109. [CrossRef] [PubMed] 205. Perycz, M.; Urbanska, A.S.; Krawczyk, P.S.; Parobczak, K.; Jaworski, J. Zipcode binding protein 1 regulates the development of dendritic arbors in hippocampal neurons. J. Neurosci. 2011,31, 5271–5285. [CrossRef] 206. Vallhov, H.; Gutzeit, C.; Johansson, S.M.; Nagy, N.; Paul, M.; Li, Q.; Friend, S.; George, T.C.; Klein, E.; Scheynius, A.; et al. Exosomes containing glycoprotein 350 released by EBV-transformed B cells selectively target B cells through CD21 and block EBV infection in vitro. J. Immunol. 2011,186, 73–82. [CrossRef] 207. Alvarez-Erviti, L.; Seow, Y.; Schapira, A.H.; Gardiner, C.; Sargent, I.L.; Wood, M.J.; Cooper, J.M. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol. Dis. 2011 ,42, 360–367. [CrossRef] 208. Wahlgren, J.; De, L.K.T.; Brisslert, M.; Vaziri Sani, F.; Telemo, E.; Sunnerhagen, P.; Valadi, H. Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic. Acids Res. 2012 ,40, e130. [CrossRef] 209. Smyth, T.; Petrova, K.; Payton, N.M.; Persaud, I.; Redzic, J.S.; Graner, M.W.; Smith-Jones, P.; Anchordoquy, T.J. Surface functionalization of exosomes using click chemistry. Bioconjug. Chem. 2014 ,25, 1777–1784. [CrossRef] 210. Robbins, P.D.; Morelli, A.E. Regulation of immune responses by extracellular vesicles. Nat. Rev. Immunol. 2014,14, 195–208. [CrossRef] 211. Di Trapani, M.; Bassi, G.; Midolo, M.; Gatti, A.; Kamga, P.T.; Cassaro, A.; Carusone, R.; Adamo, A.; Krampera, M. Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions. Sci. Rep. 2016,6, 24120. [CrossRef] 212. Lo Sicco, C.; Reverberi, D.; Balbi, C.; Ulivi, V.; Principi, E.; Pascucci, L.; Becherini, P.; Bosco, M.C.; Varesio, L.; Franzin, C.; et al. Mesenchymal Stem Cell-Derived Extracellular Vesicles as Mediators of Anti-Inflammatory Effects: Endorsement of Macrophage Polarization. Stem Cells Transl. Med. 2017 ,6, 1018–1028. [CrossRef] [PubMed] 213. Shelke, G.V.; Lasser, C.; Gho, Y.S.; Lotvall, J. Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J. Extracell. Vesicles 2014 ,3, 10. [CrossRef] [PubMed] 214. Llorente, A.; Skotland, T.; Sylvanne, T.; Kauhanen, D.; Rog, T.; Orlowski, A.; Vattulainen, I.; Ekroos, K.; Sandvig, K. Molecular lipidomics of exosomes released by PC-3 prostate cancer cells. Biochim. Biophys. Acta 2013,1831, 1302–1309. [CrossRef] [PubMed] 215. Skotland, T.; Sandvig, K.; Llorente, A. Lipids in exosomes: Current knowledge and the way forward. Prog. Lipid. Res. 2017,66, 30–41. [CrossRef] 216. Zoller, M. Exosomes in Cancer Disease. Methods Mol. Biol. 2016,1381, 111–149. [CrossRef] 217. Mateescu, B.; Kowal, E.J.; van Balkom, B.W.; Bartel, S.; Bhattacharyya, S.N.; Buzas, E.I.; Buck, A.H.; de Candia, P.; Chow, F.W.; Das, S.; et al. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA—An ISEV position paper. J. Extracell. Vesicles 2017,6, 1286095. [CrossRef] 218. Ullal, A.J.; Pisetsky, D.S.; Reich, C.F., 3rd. Use of SYTO 13, a fluorescent dye binding nucleic acids, for the detection of microparticles in in vitro systems. Cytom. A 2010,77, 294–301. [CrossRef] 219. Kim, M.; Kim, G.; Hwang, D.W.; Lee, M. Delivery of High Mobility Group Box-1 siRNA Using Brain-Targeting Exosomes for Ischemic Stroke Therapy. J. Biomed. Nanotechnol. 2019,15, 2401–2412. [CrossRef] J. Clin. Med. 2020,9, 2380 25 of 25 220. Guo, S.; Perets, N.; Betzer, O.; Ben-Shaul, S.; Sheinin, A.; Michaelevski, I.; Popovtzer, R.; Offen, D.; Levenberg, S. Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury. ACS Nano 2019,13, 10015–10028. [CrossRef] 221. Lu, Y.B.; Chen, C.X.; Huang, J.; Tian, Y.X.; Xie, X.; Yang, P.; Wu, M.; Tang, C.; Zhang, W.P. Nicotinamide phosphoribosyltransferase secreted from microglia via exosome during ischemic injury. J. Neurochem. 2019 , 150, 723–737. [CrossRef] [PubMed] 222. Aucher, A.; Rudnicka, D.; Davis, D.M. MicroRNAs transfer from human macrophages to hepato-carcinoma cells and inhibit proliferation. J. Immunol. 2013,191, 6250–6260. [CrossRef] [PubMed] 223. Schorey, J.S.; Cheng, Y.; Singh, P.P.; Smith, V.L. Exosomes and other extracellular vesicles in host-pathogen interactions. EMBO Rep. 2015,16, 24–43. [CrossRef] [PubMed] 224. Romagnoli, G.G.; Zelante, B.B.; Toniolo, P.A.; Migliori, I.K.; Barbuto, J.A. Dendritic Cell-Derived Exosomes may be a Tool for Cancer Immunotherapy by Converting Tumor Cells into Immunogenic Targets. Front. Immunol. 2014,5, 692. [CrossRef] [PubMed] 225. Kaur, S.; Singh, S.P.; Elkahloun, A.G.; Wu, W.; Abu-Asab, M.S.; Roberts, D.D. CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells. Matrix Biol. 2014 , 37, 49–59. [CrossRef] 226. Nojehdehi, S.; Soudi, S.; Hesampour, A.; Rasouli, S.; Soleimani, M.; Hashemi, S.M. Immunomodulatory effects of mesenchymal stem cell-derived exosomes on experimental type-1 autoimmune diabetes. J. Cell Biochem. 2018,119, 9433–9443. [CrossRef] 227. Jin, J.; Shi, Y.; Gong, J.; Zhao, L.; Li, Y.; He, Q.; Huang, H. Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte. Stem Cell Res. Ther. 2019,10, 95. [CrossRef] 228. Zhu, L.L.; Huang, X.; Yu, W.; Chen, H.; Chen, Y.; Dai, Y.T. Transplantation of adipose tissue-derived stem cell-derived exosomes ameliorates erectile function in diabetic rats. Andrologia 2018,50, e12871. [CrossRef] 229. Kojima, R.; Bojar, D.; Rizzi, G.; Hamri, G.C.; El-Baba, M.D.; Saxena, P.; Auslander, S.; Tan, K.R.; Fussenegger, M. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat. Commun. 2018,9, 1305. [CrossRef] [PubMed] 230. Xu, L.; Zhai, L.; Ge, Q.; Liu, Z.; Tao, R. Vacuolar Protein Sorting 4B (VPS4B) Regulates Apoptosis of Chondrocytes via p38 Mitogen-Activated Protein Kinases (MAPK) in Osteoarthritis. Inflammation 2017 ,40, 1924–1932. [CrossRef] 231. Scheffer, L.L.; Sreetama, S.C.; Sharma, N.; Medikayala, S.; Brown, K.J.; Defour, A.; Jaiswal, J.K. Mechanism of Ca(2)(+)-triggered ESCRT assembly and regulation of cell membrane repair. Nat. Commun. 2014 ,5, 5646. [CrossRef] [PubMed] 232. Dany, M.; Gencer, S.; Nganga, R.; Thomas, R.J.; Oleinik, N.; Baron, K.D.; Szulc, Z.M.; Ruvolo, P.; Kornblau, S.; Andreeff, M.; et al. Targeting FLT3-ITD signaling mediates ceramide-dependent mitophagy and attenuates drug resistance in AML. Blood 2016,128, 1944–1958. [CrossRef] 233. Ganesan, V.; Colombini, M. Regulation of ceramide channels by Bcl-2 family proteins. FEBS Lett. 2010 ,584, 2128–2134. [CrossRef] [PubMed] 234. Faure, J.; Lachenal, G.; Court, M.; Hirrlinger, J.; Chatellard-Causse, C.; Blot, B.; Grange, J.; Schoehn, G.; Goldberg, Y.; Boyer, V.; et al. Exosomes are released by cultured cortical neurones. Mol. Cell. Neurosci. 2006 , 31, 642–648. [CrossRef] 235. Kramer-Albers, E.M.; Bretz, N.; Tenzer, S.; Winterstein, C.; Mobius, W.; Berger, H.; Nave, K.A.; Schild, H.; Trotter, J. Oligodendrocytes secrete exosomes containing major myelin and stress-protective proteins: Trophic support for axons? Proteomics Clin. Appl. 2007,1, 1446–1461. [CrossRef] [PubMed] 236. Lachenal, G.; Pernet-Gallay, K.; Chivet, M.; Hemming, F.J.; Belly, A.; Bodon, G.; Blot, B.; Haase, G.; Goldberg, Y.; Sadoul, R. Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity. Mol. Cell. Neurosci. 2011,46, 409–418. [CrossRef] 237. Messenger, S.W.; Woo, S.S.; Sun, Z.; Martin, T.F.J. A Ca(2+)-stimulated exosome release pathway in cancer cells is regulated by Munc13-4. J. Cell Biol. 2018,217, 2877–2890. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).