Mesenchymal stromal cell exosome-enhanced regulatory T-cell production through an antigen-presenting cell-mediated pathway.

BACKGROUND AIMS: The immunomodulatory property of mesenchymal stromal cell (MSC) exosomes is well documented. On the basis of our previous report that MSC exosomes increased regulatory T-cell (Treg) production in mice with allogenic skin graft but not in ungrafted mice, we hypothesize that an activated immune system is key to exosome-mediated Treg production. METHODS: To test our hypothesis, MSC exosomes were incubated with mouse spleen CD4+ T cells that were activated with either anti-CD3/CD28 mAbs or allogenic antigen-presenting cell (APC)-enriched spleen CD11c+ cells to determine whether production of mouse CD4+CD25+ T cells or CD4+CD25+Foxp3+ Tregs could be induced. MSC exosomes were also administered to the lethal chimeric human-SCID mouse model of graft-versus-host disease (GVHD) in which human peripheral blood mononuclear cells were infused into irradiated NSG mice to induce GVHD. RESULTS: We report here that MSC exosome-induced production of CD4+CD25+ T cells or CD4+CD25+Foxp3+ Tregs from CD4+ T cells activated by allogeneic APC-enriched CD11C+ cells but not those activated by anti-CD3/CD28 mAbs. This induction was exosome- and APC dose-dependent. In the mouse GVHD model in which GVHD was induced by transplanted human APC-stimulated human anti-mouse CD4+ T cell effectors, MSC exosome alleviated GVHD symptoms and increased survival. Surviving exosome-treated mice had a significantly higher level of human CD4+CD25+CD127low/- Tregs than surviving mice treated with Etanercept, a tumor necrosis factor inhibitor. CONCLUSIONS: MSC exosome enhanced Treg production in vitro and in vivo through an APC-mediated pathway.

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.

Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC.

MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA.

Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles (EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.

Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles.

The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs) could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.

Mesenchymal stem cell exosomes.

MSCs are an extensively used cell type in clinical trials today. The initial rationale for their clinical testing was based on their differentiation potential. However, the lack of correlation between functional improvement and cell engraftment or differentiation at the site of injury has led to the proposal that MSCs exert their effects not through their differentiation potential but through their secreted product, more specifically, exosomes, a type of extracellular vesicle. We propose here that MSC exosomes function as an extension of MSC's biological role as tissue stromal support cells. Like their cell source, MSC exosomes help maintain tissue homeostasis for optimal tissue function. They target housekeeping biological processes that operate ubiquitously in all tissues and are critical in maintaining tissue homeostasis, enabling cells to recover critical cellular functions and begin repair and regeneration. This hypothesis provides a rationale for the therapeutic efficacy of MSCs and their secreted exosomes in a wide spectrum of diseases. Here, we give a brief introduction of the biogenesis of MSC exosomes, review their physiological functions and highlight some of their biochemical potential to illustrate how MSC exosomes could restore tissue homeostasis leading to tissue recovery and repair.

Therapeutic MSC exosomes are derived from lipid raft microdomains in the plasma membrane.

BACKGROUND: Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when purified by high performance liquid chromatography as a population of homogenously sized particles with a hydrodynamic radius of 55-65 nm reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury. As these vesicles exhibit many biophysical and biochemical properties of exosomes, they were identified as exosomes. Here we investigated if these lipid vesicles were indeed exosomes that have an endosomal biogenesis. METHOD: In most cells, endocytosis is thought to occur at specialized microdomains known as lipid rafts. To demonstrate an endosomal origin for MSC exosomes, MSCs were pulsed with ligands e.g. transferrin (Tfs) and Cholera Toxin B (CTB) that bind receptors in lipid rafts. The endocytosed ligands were then chased to determine if they were incorporated into the exosomes. RESULTS: A fraction of exogenous Tfs was found to recycle into MSC exosomes. When MSCs were pulsed with labelled Tfs in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, Tf incorporation in CD81-immunoprecipitate was reduced during the chase. CTB which binds GM1 gangliosides that are enriched in lipid rafts extracted exosome-associated proteins, CD81, CD9, Alix and Tsg101 from MSC-conditioned medium. Exogenous CTBs were pulse-chased into secreted vesicles. Extraction of Tf- or CTB-binding vesicles in an exosome preparation mutually depleted each other. Inhibition of sphingomyelinases reduced CTB-binding vesicles. CONCLUSION: Together, our data demonstrated that MSC exosomes are derived from endocytosed lipid rafts and that their protein cargo includes exosome-associated proteins CD81, CD9, Alix and Tsg101.

Mesenchymal stem cell exosome ameliorates reperfusion injury through proteomic complementation.

The therapeutic efficacy of mesenchymal stem cell (MSC) transplantation has recently been attributed to exosomes when a single bolus of MSC exosomes prior to reperfusion of ischemic myocardium ameliorates reperfusion injury and reduces infarct size. In this article we review the therapeutic efficacy of MSC exosome in ameliorating cell intrinsic factors in reperfusion injury by focusing on the proteome complementation of exosomes and reperfused myocardium. The well-documented ATP deficit and initiation of apoptosis during ischemia and reperfusion were recently found to be underpinned by a proteomic deficit in enzymes critical for fatty acid oxidation, glycolysis and tricarboxylic acid cycle, and a proteomic surplus of proapoptotic proteins. Interestingly, this deficit in glycolytic enzymes was complemented by an abundance in MSC exosomes and the surplus of proapoptotic proteins was circumvented by CD73 that could increase survival signaling through the activation of reperfusion injury salvage kinases. Together, this provides a window of opportunity for the cells to repair and regenerate thus constituting the rationale for the therapeutic efficacy of MSC exosomes.

Exosomes for drug delivery - a novel application for the mesenchymal stem cell.

Exosomes are the most extensively characterized class of secreted membrane vesicles that carry proteins and RNAs for intercellular communication. They are increasingly seen as possible alternatives to liposomes as drug delivery vehicles. Like liposomes, they could deliver their cargo across the plasma membrane and provide a barrier against premature transformation and elimination. In addition, these naturally-occurring secreted membrane vesicles are less toxic and better tolerated in the body as evidenced by their ubiquitous presence in biological fluids, and have an intrinsic homing ability. They are also amenable to in vivo and in vitro loading of therapeutic agents, and membrane modifications to enhance tissue-specific homing. Here we propose human mesenchymal stem cells as the ideal cell source of exosomes for drug delivery. Mesenchymal stem cell transplantation for various disease indications has been extensively tested and shown to be safe in numerous clinical trials. These cells are also prolific producers of immunologically inert exosomes. Immortalization of these cells does not compromise the quantity or quality of exosome production, thus enabling infinite and reproducible exosome production from a single cell clone.

Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery.

Advances in biomedical research have generated an unprecedented number of potential targets for therapeutic intervention to treat disease or delay disease progression. Unfortunately, many of these targets are not druggable as they are intracellular, present in many cell types, poorly soluble or rapidly inactivated. Although synthetic drug vehicles have successfully circumvented many of these problems, natural particulates such as exosomes that intrinsically possess many attributes of a drug delivery vehicle are highly attractive as potentially better alternatives. Of the cell types known to produce exosomes, the readily available proliferative, immunosuppressive and clinically tested human mesenchymal stem cell (MSC) is the most prolific producer. Its exosomes are therapeutic in animal model of disease and exhibit immunosuppressive activity. The quality and quantity of exosome production is not compromised by immortalization to create a permanent MSC cell line. Therefore, MSC is well suited for mass production of exosomes that are ideal for drug delivery.

High glucose predisposes gene expression and ERK phosphorylation to apoptosis and impaired glucose-stimulated insulin secretion via the cytoskeleton.

Chronic high glucose (HG) inflicts glucotoxicity on vulnerable cell types such as pancreatic ß cells and contributes to insulin resistance and impaired insulin secretion in diabetic patients. To identify HG-induced cellular aberrations that are candidate mediators of glucotoxicity in pancreatic ß cells, we analyzed gene expression in ERoSHK6, a mouse insulin-secreting cell line after chronic HG exposure (six-day exposure to 33.3 mM glucose). Chronic HG exposure which reduced glucose-stimulated insulin secretion (GSIS) increased transcript levels of 185 genes that clustered primarily in 5 processes namely cellular growth and proliferation; cell death; cellular assembly and organization; cell morphology; and cell-to-cell signaling and interaction. The former two were validated by increased apoptosis of ERoSHK6 cells after chronic HG exposure and reaffirmed the vulnerability of ß cells to glucotoxicity. The three remaining processes were partially substantiated by changes in cellular morphology and structure, and instigated an investigation of the cytoskeleton and cell-cell adhesion. These studies revealed a depolymerized actin cytoskeleton that lacked actin stress fibers anchored at vinculin-containing focal adhesion sites as well as loss of E-cadherin-mediated cell-cell adherence after exposure to chronic HG, and were concomitant with constitutive ERK1/2 phosphorylation that was refractory to serum and glucose deprivation. Although inhibition of ERK phosphorylation by PD98059 promoted actin polymerization, it increased apoptosis and GSIS impairment. These findings suggest that ERK phosphorylation is a proximate regulator of cellular processes targeted by chronic HG-induced gene expression and that dynamic actin polymerization and depolymerization is important in ß cell survival and function. Therefore, chronic HG alters gene expression and signal transduction to predispose the cytoskeleton towards apoptosis and GSIS impairment.

Delineating biological pathways unique to embryonic stem cell-derived insulin-producing cell lines from their noninsulin-producing progenitor cell lines.

To identify unique biochemical pathways in embryonic stem cell-derived insulin-producing cells as potential therapeutic targets to prevent or delay beta-cell dysfunction or death in diabetic patients, comparative genome-wide gene expression studies of recently derived mouse insulin-producing cell lines and their progenitor cell lines were performed using microarray technology. Differentially expressed genes were functionally clustered to identify important biochemical pathways in these insulin-producing cell lines. Biochemical or cellular assays were then performed to assess the relevance of these pathways to the biology of these cells. A total of 185 genes were highly expressed in the insulin-producing cell lines, and computational analysis predicted the pentose phosphate pathway (PPP), clathrin-mediated endocytosis, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway as important pathways in these cell lines. Insulin-producing ERoSHK cells were more resistant to hydrogen peroxide (H(2)O(2))-induced oxidative stress. Inhibition of PPP by dehydroepiandrosterone and 6-aminonicotinamide abrogated this H(2)O(2) resistance with a concomitant decrease in PPP activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Clathrin-mediated endocytosis, which is essential in maintaining membrane homeostasis in secreting cells, was up-regulated by glucose in ERoSHK but not in their progenitor ERoSH cells. Its inhibition by chlorpromazine at high glucose concentration was toxic to the cells. Troglitazone, a PPARG agonist, up-regulated expression of Ins1 and Ins2 but not Glut2. Gene expression analysis has identified the PPP, clathrin-mediated endocytosis, and the PPAR signaling pathway as the major delineating pathways in these insulin-producing cell lines, and their biological relevance was confirmed by biochemical and cellular assays.