Cell-free Therapeutics Research Articles

Biology of Exfoliation of Plasma Membrane-Derived Vesicles and the Radiation Response: Historical Background, Applications in Biodosimetry and Cell-Free Therapeutics, and Quantal Mechanisms for Their Release and Function with Implications for Space Travel.

This historical review of extracellular vesicles in the setting of exposure to ionizing radiation (IR) traces our understanding of how vesicles were initially examined and reported in the literature in the late 1970s (for secreted exosomes) and early 1980s (for plasma membrane-derived, exfoliated vesicles) to where we are now and where we may be headed in the next decade. An emphasis is placed on biophysical properties of extracellular vesicles, energy consumption and the role of vesiculation as an essential component of membrane turnover. The impact of intercellular signal trafficking by vesicle surface and intra-vesicular lipids, proteins, nucleic acids and metabolites is reviewed in the context of biomarkers for estimating individual radiation dose after exposure to radiation, pathogenesis of disease and development of cell-free therapeutics. Since vesicles express both growth stimulatory and inhibitory molecules, a hypothesis is proposed to consider superposition in a shared space and entanglement of molecules by energy sources that are external to human cells. Implications of this approach for travel in deep space are briefly discussed in the context of clinical disorders that have been observed after space travel.

Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling

BackgroundStem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection.MethodsEVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385.ResultsWe demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs.ConclusionsIn this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.

Activated STING-containing R-EVs from iPSC-derived MSCs promote antitumor immunity

We recently revealed that activated STING is secreted into RAB22A-induced extracellular vesicles (R-EVs) and promotes antitumor immunity in cancer cells. Whether mesenchymal stem cell (MSC)-derived R-EVs containing activated STING can be used as a novel antitumor immunotherapy remains unclear, as MSC-derived EVs are promising cell-free therapeutics due to their superior biocompatibility and safety, as well as low immunogenicity. Here, we report that induced pluripotent stem cell (iPSC)-derived MSCs can generate R-EVs with a size and mechanism of formation that are similar to those of R-EVs produced from cancer cells. Furthermore, these MSC-derived R-EVs containing activated STING induced IFNβ expression in recipient THP-1 monocytes and antitumor immunity in mice. Our findings reveal that the use of MSC-derived R-EVs containing activated STING is a promising cell-free strategy for antitumor immunity.

Pluronic F127 composite hydrogel for the repair of contraction suppressed full-thickness skin wounds in a rabbit model

Hydrogels are commonly used as carriers for cell delivery due to their similarities to the extracellular matrix. A contraction-suppressed full-thickness wound model was used to evaluate the therapeutic potential of Pluronic F127 (PF127) hydrogel loaded with adipose-derived stromal vascular fraction (AdSVF), mesenchymal stem cells (AdMSC), and conditioned media (AdMSC-CM) for the repair of wounds in a rabbit model. The experimental study was conducted on forty-eight healthy adult New Zealand white rabbits randomly divided into eight groups with six animals each and treated with AdSVF, AdMSC, and AdMSC-CM as an injectable or topical preparation. The healing potential of different adipose-derived cell-based and cell-free therapeutics was evaluated based on percentage wound healing, period of epithelialization, epidermal thickness, scar evaluation, histopathology analysis, histochemical evaluation, immunohistochemistry (collagen type I), and hydroxyproline assay by comparing with the positive and negative control. Collagen density analysis using different staining methods, immunohistochemistry, and hydroxyproline assay consistently showed that delivering AdMSC and AdMSC-CM in PF127 hydrogel enhanced epithelialization, collagen production, and organization, contributing to improved tissue strength and quality. Even though allogeneic AdSVF was found to promote wound healing in rabbits, it has a lower potential than AdMSC and AdMSC-CM. The wound healing potential of AdMSC and AdMSC-CM was enhanced when loaded in PF127 hydrogel and applied topically. Even though wounds treated with AdMSC outperformed AdMSC-CM, a significant difference in the healing quality was not observed in most instances, indicating almost similar therapeutic potential. The findings indicate that the wound healing potential of AdMSC and AdMSC-CM was enhanced when loaded in PF127 hydrogel and applied topically. These treatments promoted collagen production, tissue organization, and epidermal regeneration, ultimately improving overall healing outcomes.

Bioregenerative Applications of the Human Mesenchymal Stem Cell- Derived Secretome: Part-I

Mesenchymal stem cells hold many therapeutic benefits in treating diverse disease states, but autologous requirements, high costs, lack of standardization and other factors limit their widespread application. Additionally, researchers have discovered that many mesenchymal stem cell in-vivo benefits originate from their paracrine factors. Therefore, cell-free therapeutics, including mesenchymal stem cell-derived conditioned medium and extracellular vesicles have been suggested as alternative bioregenerative therapies. This literature review summarizes mesenchymal stem cell application, the benefits of cell-free strategies and the components of its secretome. This manuscript is a companion piece to “Part 2: Bioregenerative Applications of the Human Mesenchymal Stem Cell-Derived Secretome,” included in this issue, which contains the results of in-vivo studies applying the conditioned medium and extracellular vesicles to human and animal models, ongoing clinical trials, limitations to cell-free strategies and future directions for the wide-scale adoption of these therapies.

Pan PPAR agonist stimulation of induced MSCs produces extracellular vesicles with enhanced renoprotective effect for acute kidney injury

BackgroundAcute kidney injury (AKI) has a complex pathophysiology and imposes serious health concerns worldwide. Extracellular vesicles (EVs) derived from induced mesenchymal stem cells (iMSCs) have been recognized as novel cell-free therapeutics for various inflammatory and degenerative disorders. In this study, we investigated whether iMSCs stimulated with a pan-peroxisome proliferator-activated receptor (PPAR) agonist could enhance the therapeutic efficacy of EVs against AKI.MethodsHuman iMSCs were primed with or without lanifibranor, a PPAR agonist for 24 h, and EVs were collected after an additional 24 h. The basic characteristics of EVs were evaluated using cryo-transmission electron microscopy imaging, immunoblot detection of EV markers, nanoparticle tracking analysis, and localization in AKI kidneys. In vitro, the potential of the EVs to promote the growth and survival of HK-2 cells undergoing cisplatin-induced apoptosis and anti-inflammatory effects in M1-polarized THP-1 was compared. Subsequently, AKI was induced in BALB/c mice using cisplatin. After 8 and 24 h of cisplatin treatment, iMSC-EVs or pan-PPAR-iMSC-EVs were injected intravascularly. At 96 h after cisplatin administration, the renoprotective effects of iMSC-EVs or pan-PPAR-iMSC-EVs in inhibiting inflammation and apoptosis were compared using serum biochemistry, histology, immunohistochemistry, and gene expression analysis by qPCR.ResultsBoth EV types expressed EV markers and had typical EV morphology, and their localization in the renal tissue was confirmed. The proliferation and survival of HK-2 cells were higher in pan-PPAR-iMSC-EVs than those in iMSC-EVs. In M1-polarized THP-1 cells, the reduction in the mRNA expression of inflammatory cytokines was more significant in pan-PPAR-iMSC-EVs than that in iMSC-EVs. In the mouse model of cisplatin-induced AKI, pan-PPAR-iMSC-EVs markedly enhanced renoprotective effects compared to iMSC-EVs. Specifically, pan-PPAR-iMSC-EVs reduced tissue inflammation, immune cell infiltration, and apoptosis. Pan-PPAR-iMSC-EVs also increased renal capillary density.ConclusionPriming iMSCs with a PPAR agonist significantly improved the therapeutic potential of EVs by reducing inflammation and apoptosis. The reported strategy may contribute to the development of a novel cell-free option for AKI treatment.Trial registration: Not applicable.

Exploring the role of polymers to overcome ongoing challenges in the field of extracellular vesicles.

Extracellular vesicles (EVs) are naturally occurring nanoparticles released from all eucaryotic and procaryotic cells. While their role was formerly largely underestimated, EVs are now clearly established as key mediators of intercellular communication. Therefore, these vesicles constitute an attractive topic of study for both basic and applied research with great potential, for example, as a new class of biomarkers, as cell-free therapeutics or as drug delivery systems. However, the complexity and biological origin of EVs sometimes complicate their identification and therapeutic use. Thus, this rapidly expanding research field requires new methods and tools for the production, enrichment, detection, and therapeutic application of EVs. In this review, we have sought to explain how polymer materials actively contributed to overcome some of the limitations associated to EVs. Indeed, thanks to their infinite diversity of composition and properties, polymers can act through a variety of strategies and at different stages of EVs development. Overall, we would like to emphasize the importance of multidisciplinary research involving polymers to address persistent limitations in the field of EVs.

Extracellular vesicles derived from human ESC–MSCs target macrophage and promote anti-inflammation process, angiogenesis, and functional recovery in ACS-induced severe skeletal muscle injury

BackgroundAcute compartment syndrome (ACS) is one of the most common complications of musculoskeletal injury, leading to the necrosis and demise of skeletal muscle cells. Our previous study showed that embryonic stem cells-derived mesenchymal stem cells (ESC–MSCs) are novel therapeutics in ACS treatment. As extracellular vesicles (EVs) are rapidly gaining attention as cell-free therapeutics that have advantages over parental stem cells, the therapeutic potential and mechanisms of EVs from ESC–MSCs on ACS need to be explored.MethodIn the present study, we examined the protective effects in the experimental ACS rat model and investigated the role of macrophages in mediating these effects. Next, we used transcriptome sequencing to explore the mechanisms by which ESC–MSC-EVs regulate macrophage polarization. Furthermore, miRNA sequencing was performed on ESC–MSC-EVs to identify miRNA candidates associated with macrophage polarization.ResultsWe found that intravenous administration of ESC–MSC-EVs, given at the time of fasciotomy, significantly promotes the anti-inflammation process, angiogenesis, and functional recovery of muscle in ACS. The beneficial effects were associated with ESC–MSC-EVs affecting macrophage polarization by delivering various miRNAs which regulate NF-κB, JAK/STAT, and PI3K/AKT pathways. Our data further illustrate that ESC–MSC-EVs mainly modulate macrophage polarization via the miR-21/PTEN, miR-320a/PTEN, miR-423/NLRP3, miR-100/mTOR, and miR-26a/TLR3 axes.ConclusionTogether, our results demonstrated the beneficial effects of ESC–MSC-EVs in ACS, wherein the miRNAs present in ESC–MSC-EVs regulate the polarization of macrophages.

Multifaceted action of stem cell-derived extracellular vesicles for nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease associated with metabolic syndrome. Extracellular vesicles (EVs) are essential signaling mediators containing functional biomolecules. EVs are secreted from various cell types, and recent studies have shown that mesenchymal stem cell-derived EVs have therapeutic potential against immune and metabolic diseases. In this study, we investigated whether EVs from induced mesenchymal stem cells (iMSC-EVs) regulate AMPK signaling and lipid metabolism using cell-based studies and two different mouse models of NASH (methionine/choline-deficient diet-induced and ob/ob mice). Protein analysis revealed that iMSC-EVs carry cargo proteins with the potential to regulate lipid metabolism. iMSC-EVs inhibited free fatty acid release from adipose tissues by downregulating the activity of lipolytic genes in NASH. In addition, iMSC-EVs improved hepatic steatosis by modulating AMPK signaling, which plays essential role in metabolic homeostasis in the liver. Moreover, iMSC-EVs reduced CD36 expression, contributing to the blockade of free fatty acid transport to the liver of NASH mice. Finally, iMSC-EVs reduced inflammation, endoplasmic reticulum stress, and apoptosis while promoting hepatic regeneration of the NASH liver. In conclusion, iMSC-EVs can potentially serve as cell-free therapeutics for NASH owing to their multifaceted modality.

Adipose stromal cells bioproducts as cell-free therapies: manufacturing and therapeutic dose determine in vitro functionality

BackgroundExtracellular vesicles (EV) are considered a cell-free alternative to mesenchymal stromal cell (MSC) therapy. Numerous reports describe the efficacy of EV in conferring immunomodulation and promoting angiogenesis, yet others report these activities to be conveyed in EV-free bioproducts. We hypothesized that this discrepancy may depend either on the method of isolation or rather the relative impact of the individual bioactive components within the MSC secretome.MethodsTo answer this question, we performed an inter-laboratory study evaluating EV generated from adipose stromal cells (ASC) by either sequential ultracentrifugation (UC) or size-exclusion chromatography (SEC). The effect of both EV preparations on immunomodulation and angiogenesis in vitro was compared to that of the whole secretome and of the EV-free protein fraction after SEC isolation.ResultsIn the current study, neither the EV preparations, the secretome or the protein fraction were efficacious in inhibiting mitogen-driven T cell proliferation. However, EV generated by SEC stimulated macrophage phagocytic activity to a similar extent as the secretome. In turn, tube formation and wound healing were strongly promoted by the ASC secretome and protein fraction, but not by EV. Within the secretome/protein fraction, VEGF was identified as a potential driver of angiogenesis, and was absent in both EV preparations.ConclusionsOur data indicate that the effects of ASC on immunomodulation and angiogenesis are EV-independent. Specific ASC-EV effects need to be dissected for their use as cell-free therapeutics.

Autocrine Factors Produced by Mesenchymal Stem Cells in Response to Cell-Cell Contact Inhibition Have Anti-Tumor Properties.

Recently, mesenchymal stem cell (MSC) therapies have been questioned as MSCs are capable of both promoting and inhibiting tumorigenesis. Both MSCs and tumor cells replicate to increase their population size; however, MSCs, but not tumor cells, stop dividing when they reach confluence due to cell-cell contact inhibition and then differentiate. We hypothesized that contact inhibition results in the production of effector molecules by confluent MSCs and these effectors are capable of suppressing tumor cell growth. To test this hypothesis, we co-cultured breast cancer cells (MDA-MB-231) with either confluent or sub-confluent bone-marrow-derived MSCs (BM-MSCs); in addition, we treated various tumor cells with conditioned media (CM) obtained from either confluent or sub-confluent BM-MSCs. The results showed that the growth of tumor cells co-cultured with confluent BM-MSCs or treated with CM obtained from confluent BM-MSCs was inhibited, and this effect was significantly stronger than that seen with tumor cells co-cultured with sub-confluent BM-MSCs or CM obtained from sub-confluent BM-MSCs. Subcutaneous tumor formation was completely prevented by the inoculation of tumor cells mixed with CM. In the future, soluble anti-tumor effectors, produced by confluent MSCs, may be used as cell-free therapeutics; this approach provides a solution to current concerns associated with cell-based therapies.

Preparation of Nanoparticle-Loaded Extracellular Vesicles Using Direct Flow Filtration.

Extracellular vesicles (EVs) have shown great potential as cell-free therapeutics and biomimetic nanocarriers for drug delivery. However, the potential of EVs is limited by scalable, reproducible production and in vivo tracking after delivery. Here, we report the preparation of quercetin-iron complex nanoparticle-loaded EVs derived from a breast cancer cell line, MDA-MB-231br, using direct flow filtration. The morphology and size of the nanoparticle-loaded EVs were characterized using transmission electron microscopy and dynamic light scattering. The SDS-PAGE gel electrophoresis of those EVs showed several protein bands in the range of 20-100 kDa. The analysis of EV protein markers by a semi-quantitative antibody array confirmed the presence of several typical EV markers, such as ALIX, TSG101, CD63, and CD81. Our EV yield quantification suggested a significant yield increase in direct flow filtration compared with ultracentrifugation. Subsequently, we compared the cellular uptake behaviors of nanoparticle-loaded EVs with free nanoparticles using MDA-MB-231br cell line. Iron staining studies indicated that free nanoparticles were taken up by cells via endocytosis and localized at a certain area within the cells while uniform iron staining across cells was observed for cells treated with nanoparticle-loaded EVs. Our studies demonstrate the feasibility of using direct flow filtration for the production of nanoparticle-loaded EVs from cancer cells. The cellular uptake studies suggested the possibility of deeper penetration of the nanocarriers because the cancer cells readily took up the quercetin-iron complex nanoparticles, and then released nanoparticle-loaded EVs, which can be further delivered to regional cells.

Exosomes for angiogenesis induction in ischemic disorders.

Ischaemic disorders are leading causes of morbidity and mortality worldwide. While the current therapeutic approaches have improved life expectancy and quality of life, they are unable to "cure" ischemic diseases and instate regeneration of damaged tissues. Exosomes are a class of extracellular vesicles with an average size of 100-150 nm, secreted by many cell types and considered a potent factor of cells for paracrine effects. Since exosomes contain multiple bioactive components such as growth factors, molecular intermediates of different intracellular pathways, microRNAs and nucleic acids, they are considered as cell-free therapeutics. Besides, exosomes do not rise cell therapy concerns such as teratoma formation, alloreactivity and thrombotic events. In addition, exosomes are stored and utilized more convenient. Interestingly, exosomes could be an ideal complementary therapeutic tool for ischemic disorders. In this review, we discussed therapeutic functions of exosomes in ischemic disorders including angiogenesis induction through various mechanisms with specific attention to vascular endothelial growth factor pathway. Furthermore, different delivery routes of exosomes and different modification strategies including cell preconditioning, gene modification and bioconjugation, were highlighted. Finally, pre-clinical and clinical investigations in which exosomes were used were discussed.

Exosomes from Adipose-Derived Stem Cells Alleviate Dexamethasone-Induced Bone Loss by Regulating the Nrf2/HO-1 Axis.

The widespread use of therapeutic glucocorticoids has increased the incidences of glucocorticoid-induced osteoporosis (GIOP). Oxidative stress and mitochondrial dysfunction are major causes of GIOP; therefore, alleviation of excess oxidative stress in osteoblasts is a potential therapeutic strategy for osteoporosis. Exosomes derived from ADSCs (ADSCs-Exos), as novel cell-free therapeutics, can modulate various biological processes, such as immunomodulation, reduce oxidative damage, and promote tissue repair as well as regeneration. In this study, ADSCs-Exos restored the viability and osteogenic potential of MC3T3-E1 cells by attenuating apoptosis, oxidative damage, intracellular ROS generation, and mitochondrial dysfunction. Moreover, after pretreatment with ADSCs-Exos, Nrf2 expressions were upregulated in Dex-stimulated osteoblasts. Inhibitory assays showed that silencing Nrf2 partially eliminated the protective effects of ADSCs-Exos. The rat model assays confirmed that ADSCs-Exos alleviated the Dex-induced increase in oxidation levels, restored bone mass of the distal femur, and increased the expressions of Nrf2 and osteogenic markers in bone tissues. Thus, ADSCs-Exos alleviated apoptosis and oxidative stress by regulating Nrf2/HO-1 expressions after Dex and prevented the development of GIOP in vivo.

Generation of bioactive MSC-EVs for bone tissue regeneration by tauroursodeoxycholic acid treatment.

Extracellular vesicles (EVs) are nano-sized carriers that reflect the parent cell's information and are known to mediate cell-cell communication. In order to overcome the disadvantages of mesenchymal stem cells (MSCs) in cell therapy, such as unexpected differentiation leading to tumorization, immune rejection, and other side effects, EVs derived from MSCs (MSC-EVs) with the tissue regenerative function have been studied as new cell-free therapeutics. However, therapeutic applications of EVs require overcoming several challenges. First, the production efficiency of MSC-EVs should be increased at least as much as the quantity of them are required to their clinical application; second, MSC-EVs needs to show various functionality further, thereby increasing tissue regeneration efficiency. In this study, we treated tauroursodeoxycholic acid (TUDCA), a biological derivative known to regulate cholesterol, to MSCs and investigated whether TUDCA treatment would be able to increase EV production efficiency and tissue regenerative capacity of EVs. Indeed, it appears that TUDCA priming to MSC increases the yield of MSC-EVs >2 times by reducing the cellular cholesterol level in MSCs and increasing the exocytosis-related CAV1 expression. Interestingly, it was found that the EVs derived from TUDCA-primed MSCs (T-EV) contained higher amounts of anti-inflammatory cytokines (IL1RN, IL6, IL10, and IL11) and osteogenic proteins (ALP, RUNX2, BMP2, BMPR1, and BMPR2) than those in control MSC-EVs (C-EV). Besides, it was shown that T-EV not only regulated M1/M2 macrophages differentiation of monocytes, also effectively increased the osteogenic differentiation of MSCs as well as bone tissue regeneration in a bone defect rat model. Based on these results, it is concluded that TUDCA treatment to MSC as a new approach endows EV with high-yield production and functionality. Thus, we strongly believe T-EV would be a powerful therapeutic material for bone tissue regeneration and potentially could be expanded to other types of tissue regeneration for clinical applications.

Cardiac regeneration: Options for repairing the injured heart.

Cardiac regeneration is one of the grand challenges in repairing injured human hearts. Numerous studies of signaling pathways and metabolism on cardiac development and disease pave the way for endogenous cardiomyocyte regeneration. New drug delivery approaches, high-throughput screening, as well as novel therapeutic compounds combined with gene editing will facilitate the development of potential cell-free therapeutics. In parallel, progress has been made in the field of cell-based therapies. Transplantation of human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) can partially rescue the myocardial defects caused by cardiomyocyte loss in large animals. In this review, we summarize current cell-based and cell-free regenerative therapies, discuss the importance of cardiomyocyte maturation in cardiac regenerative medicine, and envision new ways of regeneration for the injured heart.

Human Umbilical Cord Mesenchymal Stem Cells-Derived Small Extracellular Vesicles Can Be Considered as Cell-Free Therapeutics for Angiogenesis Promotion.

Angiogenesis has critical roles in several physiological processes. Restoring angiogenesis in some pathological conditions such as a few vascular diseases can be a therapeutic approach to controlling this issue. Mesenchymal stem cells (MSCs) secrete specific intracellular products known as extracellular vesicles (EVs) with high therapeutic potential which compared to their source cells, do not have the limitations of cell therapy. The angiogenic effect of the human umbilical cord MSCs (hUCMSCs)-derived small EVs are evaluated in the present work. Aim of this research is to show that hUCMSCs-derived small EVs cause differentiation of genes involved in angiogenesis like FGFR-1, FGF, VEGF, and VEGFR-2. In this experimental study, MSCs were isolated from the human umbilical cord, and after confirming their identities, their secreted EVs (including exosomes) were extracted by ultracentrifugation. The isolated small EVs were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), bicinchoninic acid assay (BCA), and Western Blotting. Then, the human umbilical vein endothelial cells (HUVECs) were treated with derived small EVs for 72 hours, and the expression of the angiogenic factors including FGFR-1, FGF, VEGF, and VEGFR-2 was evaluated by quantitative real-time-polymerase chain reaction (qPCR). Angiogenesis was also evaluated via a tube formation assay. The results demonstrated that FGFR-1, FGF, VEGF, and VEGFR-2 could be elevated 2, 2, 3.5, and 2 times, respectively, in EVs treated HUVECs, and derivative EVs can encourage tube formation in HUVECs. These findings imply that hUCMSCs-derived small EVs are valuable resources in promoting angiogenesis and are very promising in cell-free therapy.

Exosomal MicroRNAs as Novel Cell-Free Therapeutics in Tissue Engineering and Regenerative Medicine.

Extracellular vesicles (EVs) are membrane-bound vesicles (50–1000 nm) that can be secreted by all cell types. Microvesicles and exosomes are the major subsets of EVs that exhibit the cell–cell communications and pathological functions of human tissues, and their therapeutic potentials. To further understand and engineer EVs for cell-free therapy, current developments in EV biogenesis and secretion pathways are discussed to illustrate the remaining gaps in EV biology. Specifically, microRNAs (miRs), as a major EV cargo that exert promising therapeutic results, are discussed in the context of biological origins, sorting and packing, and preclinical applications in disease progression and treatments. Moreover, advanced detection and engineering strategies for exosomal miRs are also reviewed. This article provides sufficient information and knowledge for the future design of EVs with specific miRs or protein cargos in tissue repair and regeneration.

ADSCs-derived exosomes ameliorate hepatic fibrosis by suppressing stellate cell activation and remodeling hepatocellular glutamine synthetase-mediated glutamine and ammonia homeostasis

BackgroundHepatic fibrosis is a common pathologic stage in chronic liver disease development, which might ultimately lead to liver cirrhosis. Accumulating evidence suggests that adipose-derived stromal cells (ADSCs)-based therapies show excellent therapeutic potential in liver injury disease owing to its superior properties, including tissue repair ability and immunomodulation effect. However, cell-based therapy still limits to several problems, such as engraftment efficiency and immunoreaction, which impede the ADSCs-based therapeutics development. So, ADSCs-derived extracellular vesicles (EVs), especially for exosomes (ADSC-EXO), emerge as a promise cell-free therapeutics to ameliorate liver fibrosis. The effect and underlying mechanisms of ADSC-EXO in liver fibrosis remains blurred.MethodsHepatic fibrosis murine model was established by intraperitoneal sequential injecting the diethylnitrosamine (DEN) for two weeks and then carbon tetrachloride (CCl4) for six weeks. Subsequently, hepatic fibrosis mice were administrated with ADSC-EXO (10 μg/g) or PBS through tail vein infusion for three times in two weeks. To evaluate the anti-fibrotic capacity of ADSC-EXO, we detected liver morphology by histopathological examination, ECM deposition by serology test and Sirius Red staining, profibrogenic markers by qRT-PCR assay. LX-2 cells treated with TGF-β (10 ng/ml) for 12 h were conducted for evaluating ADSC-EXO effect on activated hepatic stellate cells (HSCs). RNA-seq was performed for further analysis of the underlying regulatory mechanisms of ADSC-EXO in liver fibrosis.ResultsIn this study, we obtained isolated ADSCs, collected and separated ADSCs-derived exosomes. We found that ADSC-EXO treatment could efficiently ameliorate DEN/CCl4-induced hepatic fibrosis by improving mice liver function and lessening hepatic ECM deposition. Moreover, ADSC-EXO intervention could reverse profibrogenic phenotypes both in vivo and in vitro, including HSCs activation depressed and profibrogenic markers inhibition. Additionally, RNA-seq analysis further determined that decreased glutamine synthetase (Glul) of perivenous hepatocytes in hepatic fibrosis mice could be dramatically up-regulated by ADSC-EXO treatment; meanwhile, glutamine and ammonia metabolism-associated key enzyme OAT was up-regulated and GLS2 was down-regulated by ADSC-EXO treatment in mice liver. In addition, glutamine synthetase inhibitor would erase ADSC-EXO therapeutic effect on hepatic fibrosis.ConclusionsThese findings demonstrated that ADSC-derived exosomes could efficiently alleviate hepatic fibrosis by suppressing HSCs activation and remodeling glutamine and ammonia metabolism mediated by hepatocellular glutamine synthetase, which might be a novel and promising anti-fibrotic therapeutics for hepatic fibrosis disease.

Nano-Messengers of the Heart: Promising Theranostic Candidates for Cardiovascular Maladies.

Till date, cardiovascular diseases remain a leading cause of morbidity and mortality across the globe. Several commonly used treatment methods are unable to offer safety from future complications and longevity to the patients. Therefore, better and more effective treatment measures are needed. A potential cutting-edge technology comprises stem cell-derived exosomes. These nanobodies secreted by cells are intended to transfer molecular cargo to other cells for the establishment of intercellular communication and homeostasis. They carry DNA, RNA, lipids, and proteins; many of these molecules are of diagnostic and therapeutic potential. Several stem cell exosomal derivatives have been found to mimic the cardioprotective attributes of their parent stem cells, thus holding the potential to act analogous to stem cell therapies. Their translational value remains high as they have minimal immunogenicity, toxicity, and teratogenicity. The current review highlights the potential of various stem cell exosomes in cardiac repair, emphasizing the recent advancements made in the development of cell-free therapeutics, particularly as biomarkers and as carriers of therapeutic molecules. With the use of genetic engineering and biomimetics, the field of exosome research for heart treatment is expected to solve various theranostic requirements in the field paving its way to the clinics.