Mesenchymal Stem/Stromal Cell-Derived Exosomes Part 1

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Abstract: Exosomes are nano-sized vesicles that serve as mediators for cell-to-cell communication. With their unique nucleic acids, proteins, and lipids cargo compositions that reflect the characteristics of producer cells, exosomes can be utilized as cell-free therapeutics. Among exosomes derived from various cellular origins, mesenchymal stem cell-derived exosomes (MSC-exosomes) have gained great attention due to their immunomodulatory and regenerative functions. Indeed, many studies have shown anti-inflammatory, anti-aging, and wound healing effects of MSC-exosomes in various in vitro and in vivo models. In addition, recent advances in the field of exosome biology have enabled the development of specific guidelines and quality control methods, which will ultimately lead to the clinical application of exosomes. This review highlights recent studies that investigate the therapeutic potential of MSC exosomes and relevant mode of action for skin diseases, as well as quality control measures required for the development of exosome-derived therapeutics.

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Keywords: anti-aging; anti-inflammation; hair growth; immunomodulation; mesenchymal stem cells (MSCs); MSC-exosomes; skin barrier; therapeutics; regenerative aesthetics; wound healing

1. Introduction

The discovery of extracellular vesicles(EVs)or exosomes goes back to the 1940s, and these tiny vesicles were ignored as cellular garbage bins for a long time [1-3]. They only began to draw significant attention around the mid-2000s after the re-discovery of exosomes as messengers for cell-to-cell communications [1,4-6]. It is no exaggeration to say that we are at the dawn of the exosome era. There were more than three thousand publications on EVs or exosomes and related subjects in PubMed annually in 2018 and 2019[1]. cistanche tubulosa extract The race toward the commercialization of exosome-based therapeutics has already begun [7-10]. The top four exosome start-up companies, Codiak Biosciences, Exosome Diagnostics, Evox Therapeutics, and ExoCoBio have received approximately $386.2 million in investor funding [8]. In addition, several big deals have been made between exosome start-ups and big pharma companies [10].

Exosomes are nano-sized extracellular vesicles (EVs)released by almost all eukaryotic cells [11]. In general, their size ranges from 30 nM to 200 nM. Two other subpopulations of EVs are microvesicles (100-1000 nM) and apoptotic bodies (500-2000 nM)[12-14]. Exosomes derived from stem cells have attractive therapeutic potential in several aspects [15]. It has been established that the mode of action (MoA) for therapeutic effects of stem cells is mainly paracrine effects mediated by secreted factors from stem cells [6,16]. Among parts of the secretome of stem cells, exosomes have been reported to play a major role in the paracrine effects [16-18]. Mesenchymal stem/stromal cells (MSCs) are the most preferable source of therapeutic exosomes since MSCs themselves appear to be safe based on a huge amount of clinical data over the last decade [15]. In addition, MSC-derived exosomes (MSC-exosomes)can be sterilized by filtration and produced as an off-the-shelf product, while MSCs themselves cannot. Moreover, MSC-exosomes are considered to be free from the safety issues in the context of cell-based therapy, such as tumorigenic potential by cell administration [19,20]. cistanche tubulosa reviews Indeed, MSC-exosomes have been applied as alternatives to MSCs for new cell-free therapeutic strategies in a variety of disease models including neurological, cardiovascular, immune, renal, musculoskeletal, liver, respiratory, eye, and skin diseases, as well as cancers [15,17,19,21,22].

2. MSCs as Sources of Exosomes

MSCs have both self-renewal capabilities (ie., they can generate more MSCs themselves) and differentiation (into other types of cells) potentials [23]. MSCs can be obtained from a range of tissues and body fluids, such as adipose tissue, bone marrow(BM), dental pulp, synovial fluid (SF), amniotic fluid(AF), placenta(PL), umbilical cord (UC), umbilical cord blood (UCB), and Wharton's jelly (WJ) [24]. MSCs can also be derived from embryonic stem cells(ESCs) or induced pluripotent stem cells (iPSCs) [25-27]. MSCs, depending on their origins, are able to differentiate into diverse types of cells including adipocytes, chondrocytes, osteoblasts, and myocytes [28]. In addition, MSCs have immunomodulatory properties to regulate various cells involved in immune responses, such as dendritic cells (DCs), lymphocytes, macrophages, mast cells, neutrophils, and natural killer (NK)cells [24]. On these bases, MSCs have been spotlighted as potent cell therapeutics for various diseases over the last decades.

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In the reported preclinical studies of MSC-exosomes, MSCs were isolated from various tissues/cells in the following order: BM(51%), umbilical/placental tissues(23%),adipose tissue(13%), derived from ESCs or iPSCs (8%), and others (5%)[29]. Since the characteristics and functionality of MSCs depend on their origins, it is obvious that those of MSC exosomes vary according to the origin of MSCs. However, comparative studies of MSC-exosomes by their tissue origin are still limited, and only a few reports have compared different MSC-exosomes within the same study (Table 1)[30-35]:(1)human adipose tissue-derived MSC(ASC)-exosomes exhibited a higher activity of neprilysin, an amyloid β (Aβ)peptide degrading enzyme in the brain, then human bone marrow MSC (BM-MSC)-exosomes, suggesting the therapeutic relevance of ASC-exosomes in Alzheimer's disease [30];(2) human BM-MSC-EVs and Wharton'sjelly MSC(WJ-MSC)-EVs decreased cell proliferation and induced apoptosis, while ASC-EVs increased cell proliferation and had no apoptotic effect in U87MG glioblastoma cells [31]. However, the effects of MSC-exosomes on cancer cells are controversial [36]. For example, ASC-exosomes have been reported to have anti-cancer activity on prostate cancer both in vitro and in vivo[37];(3) human menstrual fluid MSC(MSC)-exosomes and BM-MSC-exosomes promoted neurite growth both in cortical and sensory neurons, while human chorion MSC-exosomes and UC-MSC-exosomes did not. cistanche UK This suggests that an appropriate selection of MSC sources might be essential for the treatment of neurodegenerative diseases [32];(4)human iPSC MSC (MSC)-exosomes and synovial membrane MSC (SM-MSC)-exosomes both attenuated osteoarthritis(OA) in a murine model, but MSC-exosomes had a superior therapeutic effect compared to SM-MSC-exosomes [33];(5)a study comparing

canine MSCs reported that BM-MSCs released a higher level of secretome, including exosomes than ASCs did [34]; and(6) human amniotic fluid MSCs(AF-MSCs) released a higher amount of exosomes than BM-MSCs [35]. However, it is difficult to directly compare the results between the above studies, since they were not performed with comparable processes or methods for isolation, characterization, and efficacy evaluation for exosomes. In addition, variations from different donors or preparation methods for MSCs remain a prominent challenge [38,39]. Nevertheless, it is suggested that MSC-exosomes might exhibit different properties and efficacies depending on the origin of MSCs. Therefore, biological differences such as the origin of MSCs and the efficacy of their exosomes should be considered for specific clinical applications.

3. Quality Control of EVs for Development of Therapeutic EVs

It is important to manufacture clinical-grade EVs with a good manufacturing practice (GMP)-compliant process and quality control(QC)for the development of EV-based therapeutics[40-42]. Appropriate QCis also crucial for reproducible studies in academic settings. Recently, the International Society for Extracellular Vesicles(ISEV) proposed a series of the Minimal Information for Studies of Extracellular Vesicles (MISEV), finalized as MISEV2018[43-45]. The Korea Ministry of Food and Drug Safety (MFDS) published the world's first guideline for EV therapy products, entitled the Guideline on Quality, Non-clinical, and Clinical Assessment of Extracellular Vesicles Therapy Products [46. As shown in Table 2, most of the criteria in these guidelines are similar[1] and have been already been applied in GMP settings [42, A7,48]. Routine QC criteria include the determination of the quantity, size, identity, and purity of EVs.

Since these methods cannot differentiate EVs from non-EV particles, it is recommended to compare results from these methods with results from TEM, AFM, or other microscopic observations.2 Comparison with results from quantification methods such as protein quantification is also recommended. Abbreviations: AF4, multi-angle light scattering coupled to asymmetric flow field-flow fractionation; AFM, atomic force microscopy; DLS, dynamic light scattering; FCM, flow cytometry; FCS, fluorescence correlation spectroscopy; ISEV, International Society for Extracellular Vesicles; LAL, Limulus amebocyte lysate; MoA, mode of action; MFDS, Ministry of Food and Drug Safety; NTA, nanoparticle tracking analysis; RPS, resistive pulse sensing; WB, Western blotting.

3.1.EVQuantity and Size

Both the MISEV2018 and the MFDS guidelines recommend using at least two different methods for determining the quantity of EVs [45,46]. Quantification of EVs can be achieved by measuring the total amounts of proteins, lipids, or RNAs since EVs consist of all these molecules. These methods, however, do not provide information on the number of EV particles. Several methods are available to measure the number and size of particles, including nanoparticle tracking analysis (NTA), resistive pulse sensing (RPS), and dynamic light scattering (DLS). The most widely used method is NTA [42,47-53].NTA determines the number and size of particles by tracking the Brownian motion of single particles in an aqueous solution [54]. However, NTA suffers from a low resolution of poly-dispersed samples and high variations, such as inter-device, inter-assay, and intra-and inter-individual variations [55-57]. In addition, NTA does not differentiate EVs from other nanoparticles such as protein aggregates. cistanche wirkung Recently, instruments for fluorescence NTA have been introduced to detect fluorescently labeled EVs with specific antibodies [58]. Quantification of EVs, however, remains extremely challenging. New technologies and instruments have been introduced annually, especially during the ISEV conference, such as nanoflow cytometry 59,60], direct stochastic optical reconstruction microscopy [61], ExoCounter with the optical disc technology [62], and imaging flow cytometry [63]. Although it will take some time to develop fully GMP-compatible instruments, the great strides forward in methodologies for the quantification of EVs are expected to result in the overcoming of current hurdles in the near future.

3.2. EV Identity

A variety of proteins have been reported to be associated with EV, especially exosomes, including tetraspanins(CD9, CD63, and CD81), Annexins, Flotillin, and ALG-2-interacting protein X(Alix), and tumor susceptibility gene 101(TSG101)protein [45,64]. Proteins such as CD9, CD63, CD81, TSG101, and Alix are recommended as specific markers for exosomes since they are known to be highly enriched in exosomes compared to the originating cells [45,64-66]. In addition, because Alix and TSG101 are involved in the formation of multivesicular bodies (MVBs), the presence of these proteins is essential to support the endocytic origin of exosomes |43,45,64]. For QC, at least semi-quantitative methods are recommended to detect these proteins in exosomes [46]. The enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis are each suitable for both GMP-compliant facilities and general academic labs. Although Western blotting has been widely used in academic labs, this method is limited by the lack of appropriate quantification and method validation [67].

3.3.EV Purity

The purity of EVs is also a critical criterion for QC. A simple method to monitor the purity of EVs is to determine the particle-to-protein, protein-to-lipid, or RNA-to-particle ratios [45]. The absence of intracellular proteins, such as histones, lamin A/C, GRP94(i.e., HSP90B1), GM130 (i.e., GOLGA2), and cytochrome C(i.e., CYC1), is another important criterion to determine the purity of EVsorexosomes since these proteins are not enriched in exosomes due to their strict cellular localization [43,45]. Impurities from the cell culture process including antibiotics and serum should also be analyzed to monitor the removal of potentially hazardous substances [46]. Every batch of EVs should be qualified by routine QC before being used for therapeutic purposes or functional assays, even in the academic labs, to ensure reproducibility.

3.4.PotenCy Assays

Potency assays are the most important OC criterion to predict the efficacy of EVs in vivo. Regulatory authorities such as the US Food and Drug Administration (FDA) recommend using appropriate potency tests for cellular and gene therapy products [68]. The MISEV2018 and the MFDS guidelines also recommend including potency assays for EV QC [45,46]. Potency is defined as"the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result"[68]. Many biological and biochemical assays have been reported to demonstrate the potency of EVs or exosomes [69,70]. Since quantification of EVs remains challenging, the establishment of an appropriate potency assay would be an invaluable tool to monitor batch-to-batch consistency and determine the dose of EVs [71]. Although ideal potency assays should represent the MoA, it is difficult to set up an appropriate potency assay with single biochemical or isolated cell-based assays due to the difficulty in the identification of single bioactive substances in the complex cargo of EVs. As an example, it is hard to mimic the complex immune responses in vivo with in vitro cell-based assays [70-73].

4. Anti-Inflammation and Immunomodulation by MSC-Exosomes

Immune cells secrete soluble factors such as inflammatory cytokines and mediators, which can contribute in the event of inflammation [74,75]. In particular, pro-inflammatory cytokines, including tumor necrosis factor (TNF)-αx, interleukin(IL)-6, and IL-1β, are mainly produced by activated macrophages. These cytokines play important roles in the upregulation of inflammatory responses such as activation of macrophages and recruitment of additional immune cells [74,75]. In contrast, anti-inflammatory cytokines are produced by regulatory T cells (Tregs), helper T (Th)2 cells, alternatively activated macrophages, and monocytes, which control the inflammatory responses and immunity 75,76]. Major anti-inflammatory cytokines include lL-1 receptor agonist(lL-1RA), lL-4, IL-10, and transforming growth factor(TGF)-β [76]. citrus bioflavonoids These cytokines inhibit the Th1l responses and production of pro-inflammatory cytokines [76].

Inflammation is a mechanism of innate immunity in response to harmful stimuli, including pathogens, damaged cells, or irritants, and typically manifests as heat, pain, redness, swelling, and loss of function [77]. Uncontrolled chronic inflammatory responses are associated with diverse inflammatory diseases such as allergy, asthma, autoimmune diseases, inflammatory bowel disease (IBD), OA, atherosclerosis, and hepatitis [77-79]. In addition, many scientists now consider inflammation as the root cause of most chronic diseases such as heart attacks, strokes, type 2 diabetes, Alzheimer's disease, and even cancer [80,81]. Therefore, regulation of inflammation is an important therapeutic target to treat inflammatory diseases. It has been demonstrated that MSCs have the property of intrinsic immunosuppressive capabilities to alleviate inflammation and immune responses [82]. MSC-exosomes can be an excellent alternative to MSC cell therapy since MSC-exosomes possess similar biological functions to the originating cells, while they are more stable and have lower immunogenicity compared to their originating cells [83]. In fact, anti-inflammatory and immunomodulatory functions of MSC-exosomes have been extensively reported (Table 3) [21,84-151].

4.1.Macrophage Polarization

There is accumulating evidence that MSC-exosomes promotes macrophage polarization from M1 toward M2. Ml macrophages are characterized by the expression of a broad spectrum of pro-inflammatory cytokines and chemokines, such as IL-1β, IL-12, and TNF-α. By contrast, the M2 macrophage phenotype is induced by Th2 cytokines and leads to the secretion of anti-inflammatory factors, such as IL-10 and TGF-β, and M2 markers such as IL-1RA, CD163, and C-C motif chemokine 22 (CCL22)[152]. It has been reported that human BM-MSC-exosomes and jaw bone marrow MSC (JM-MSC)-exosomes promote cutaneous wound healing [86], and ameliorate bronchopulmonary dysplasia(BPD)[86] through macrophage M2 polarization. The miR-223 contained in exosomes alleviated inflammation and accelerated wound healing by inducing macrophage M2 polarization. Co-culture with BM-MSC-exosomes increased the expression of miR-223 and decreased the expression of PBX/knotted homeobox 1(PKNOX1) protein, an important regulator of macrophage polarization, in macrophages isolated from peripheral blood mononucleated cells (PBMCs). Besides, after co-culture with BM-MSC-exosomes CD206-positive macrophages were elevated, and miR-223 inhibitors reversed this elevation [85]. In a high-fat diet (HFD) mouse model, miR-223 deficiency enhanced infiltration of M1 macrophage, and increased production of pro-inflammatory cytokines, but decreased M2-associated biomarkers including peroxisome proliferator-activated receptor(PPARy)and arginase 1(ARG1)[153]. Another study elucidated that human UC-MSC-exosomes also promote M2 macrophage activation and regulate diabetic cutaneous wound healing [87]. Compared to those from unconditioned UC-MSCs, exosomes from LPS-preconditioned UC-MSCs contained a high level of let-7b, ameliorated inflammation, and promoted wound healing more intensely. UC-MSC-exosomes decreased toll-like receptor 4(TLR4) and phospho (p)-p65 proteins regardless of LPS preconditioning. After treatment of LPS-preconditioned UC-MSC-exosomes, ARG1, an M2 macrophage marker, was increased, and inducible nitric oxide synthase (iNOS), an M1 macrophage marker, was decreased [88]. The let-7b targets TLR4, activation of which leads to activation of nuclear factor-kB(NF-kB).Additionally,let-7b downregulates the expression of cyclooxygenase-2(COX-2) and cyclin D1 proteins [154]. It was revealed that UC-MSC-exosomes suppress inflammation and promote wound healing by inducing secretion of cytokines from M2 macrophages in rats with severe burn-induced skin inflammation through downregulation of TLR4, NF-KB, and p-p65 expression [89]. A higher level of miR-181c was observed in UC-MSC-exosomes compared to in human dermal fibroblast (HDF)-exosomes. The expression level of miR-181c was decreased by burn injury and was increased after treatment of UC-MSC-exosomes in the cutaneous wound. In addition, treatment of UC-MSC-exosomes reduced the expression of TNF-α and IL-1β and increased the expression of IL-10. These effects were strengthened by exosomes derived from miR-181c-overexpressed UC-MSCs [88]. In an experiment conducted in mouse astrocytes, the expression level of miR-181c was decreased by LPS, a TLR4 receptor ligand. Overexpression of miR-181c increased IL-10 secretion induced by LPS[155]. In primary microglia, oxygen-glucose deprivation(OGD)upregulated TLR4, while miR-181c reversed this upregulation. The miR-181c also downregulated NF-kB and pro-inflammatory cytokines such as TNF-α, IL-1β, and iNOS induced by OGD[156]. In addition, it was found that human MSC-exosomes induced macrophage M2 polarization, which was confirmed by the increased ARG1/iNOS ratio, which led to the alleviation of inflammation in the diabetic cutaneous wound [89].

Moreover, exosomes derived from various MSCs also play an important role in promoting the activation of M2 macrophages in other inflammatory diseases as well as cutaneous wounds. It was found that mouse BM-MSC-exosomes relieve inflammation in atherosclerosis via macrophage M2 polarization in vivo through the let-7/high mobility group AT-Hook 2(HMGA2)/NF-kB pathway [90]. Enrichment of the let-7 family was found in BM-MSC-exosomes, and treatment of BM-MSC-exosomes upregulated the let-7level in ApoE-/-mice [90]. Zhao et al.revealed that mouse BM-MSC-exosomes also attenuated myocardial ischemia-reperfusion (IR) injury through polarizing macrophages toward M2 phenotypes(iNOS-CD206+), and increasing IL-10 and ARG1, which are regulated by miR-182 targeting TLR4[91]. Human BM-MSC-exosomes has been reported to reduce dextran sodium sulfate (DSS)-induced IBD in mice through the polarization of M2b macrophages in a metallothionein-2 (MT2A)-dependent manner [92]. Another report revealed that mouse ESC-exosomes improved cardiomyopathy by increasing M2 macrophages and IL-10 release [157]. Additionally, it was reported that rat ASC-exosomes ameliorated myocardial infarction by promoting M2 macrophage polarization, which is regulated by increasing sphingosine-1-phosphate receptor 1(S1PR1)[93]. The importance of the sphingosine 1-phosphate (S1P)/sphingosine kinase 1 (SphK1)/S1PR axis was further confirmed by silencing of S1PR1, which abolished the decrease of hypoxia-induced apoptosis by ASC-exosomes in H9c2 cells. Similarly, human ASC-exosomes induced M2 macrophage markers in human PBMCs [94]. Heo et al. revealed that human ASC-exosomes also induce the M2 macrophage phenotype by confirming the increased level of transcription factors (e.g., signal transducer and activator of transcription 6 (STAT6), MAF BZIP transcription factor B (MafB), etc.), which led to regulating immunomodulatory and anti-inflammatory effects such as increased Tregs and anti-inflammatory cytokines (e.g., IL-10 and TNF-α-stimulated gene-6(TSG-6))[94]. Mouse ASC-exosomes also induced M2 macrophage polarization and reduced inflammation of white adipose tissues(WAT)in obese mice[96]. These effects are dependent on a transcription factor, STAT3, in ASC-exosomes. Furthermore, ASC-exosome-educated M2 macrophages induced proliferation of ASCs themselves and production of lactate from ASCs, which further promoted WAT beiging [95]. However, further studies are needed to understand the detailed underlying molecular mechanism for the regulation of M2 macrophage polarization by MSC-exosomes.

This article is extracted from Cells 2020, 9, 1157; doi:10.3390/cells9051157 www.mdpi.com/journal/cells