Human Exosomes Research Articles

Human placental exosomes induce maternal systemic immune tolerance by reprogramming circulating monocytes

BackgroundThe maternal immune system needs to tolerate the semi-allogeneic fetus in pregnancy. The adaptation occurs locally at the maternal–fetal interface as well as systemically through the maternal circulation. Failure to tolerate the paternal antigens may result in pregnancy complications, such as pregnancy loss and pre-eclampsia. However, the mechanism that regulates maternal immune tolerance, especially at the systemic level, is still an enigma. Here we report that the first-trimester placenta-derived exosomes (pEXOs) contribute to maternal immune tolerance by reprogramming the circulating monocytes.ResultspEXOs predominantly target monocytes and pEXO-educated monocytes exhibit an immunosuppressive phenotype as demonstrated by reduced expression of marker genes for monocyte activation, T-cell activation and antigen-process/presentation at the transcriptomic level. They also have a greater propensity towards M2 polarization when compared to the monocytes without pEXO treatment. The inclusion of pEXOs in a monocyte-T-cell coculture model significantly reduces proliferation of the T helper cells and cytotoxic T cells and elevates the expansion of regulatory T cells. By integrating the microRNAome of pEXO and the transcriptomes of pEXO-educated monocytes as well as various immune cell functional assays, we demonstrate that the pEXO-derived microRNA miR-29a-3p promotes the expression of programmed cell death ligand-1, a well-known surface receptor that suppresses the adaptive immune system, by down-regulation of phosphatase and tensin homolog in monocytes.ConclusionsThis is the first report to show how human pEXO directly regulates monocyte functions and its molecular mechanism during early pregnancy. The results uncover the importance of pEXO in regulating the maternal systemic immune response during early pregnancy by reprogramming circulating monocytes. The study provides the basis for understanding the regulation of maternal immune tolerance to the fetal allograft.Graphical

Human adipose mesenchymal stem cells modulate inflammation and angiogenesis through exosomes

Stem cell-derived exosomes are efficient and safe therapeutic tools for transferring endogenous biological cargo or functional biomolecules for regenerative medicine. The regulation of inflammation and angiogenesis plays a pivotal role in wound healing and tissue regeneration. The purpose of this study was to investigate the anti-inflammatory and pro-angiogenic roles of human adipose mesenchymal stem cell-derived exosomes, focusing on the underlying mechanisms. Exosomes inhibited LPS-induced inflammation by activating ROCK1 and PTEN expression. Moreover, microRNAs (miR-132 and miR-146a) released from exosomes upregulated the expression of pro-angiogenic genes and promoted proliferation activity and tube formation in human umbilical vein endothelial cells. Exosomal effects were verified using ROCK1/PTEN inhibitors for anti-inflammation and miR-132/miR-146a inhibitors for pro-angiogenesis. Our findings suggest that exosomes exert anti-inflammatory effects by targeting the ROCK1/PTEN pathway and exhibit pro-angiogenic effects via delivery of miR-132 and miR-146a. Taken together, these results suggest that exosomes may be promising therapeutic candidates for curing diseases involved in inflammation and angiogenesis.

Mesenchymal Stem Cell Exosomes Promote Functional Osteochondral Repair in a Clinically Relevant Porcine Model

Background: Previous studies have reported the efficacy of human mesenchymal stem cell (MSC) exosomes for the repair of osteochondral defects in rats and rabbits. However, the safety and efficacy of MSC exosomes remain to be validated in a clinically relevant large animal model. Purpose: To validate the safety and efficacy of human MSC exosomes for osteochondral repair in a clinically relevant micropig model. Study Design: Controlled laboratory study. Methods: Bilateral osteochondral defects (6-mm diameter and 1-mm depth) were surgically created in the medial femoral condyles in knees of 12 micropigs. The pigs then received 2-mL intra-articular injections of MSC exosomes and hyaluronic acid (HA) (Exosome+HA) or HA alone after surgery and thereafter at 8 and 15 days. Osteochondral repair was assessed by magnetic resonance imaging (MRI) at 15 days and at 2 and 4 months after surgery as well as by macroscopic, histological, biomechanical, and micro—computed tomography (micro-CT) analyses at 4 months after surgery. Results: Exosome+HA–treated defects demonstrated significantly better MRI scores than HA-treated defects at 15 days and at 2 and 4 months. Additionally, Exosome+HA–treated defects demonstrated functional cartilage and subchondral bone repair, with significantly better macroscopic and histological scores and biomechanical properties (Young modulus and stiffness) than HA-treated defects at 4 months. Micro-CT further showed significantly higher bone volume and trabecular thickness in the subchondral bone of Exosome+HA–treated defects than that of HA-treated defects. Importantly, no adverse response or major systemic alteration was observed in any of the animals. Conclusion: This study shows that the combination of MSC exosomes and HA administered at a clinically acceptable frequency of 3 weekly intra-articular injections can promote functional cartilage and subchondral bone repair, with significantly improved morphological, histological, and biomechanical outcomes in a clinically relevant porcine model. Clinical Relevance: Our findings provide a robust scientific rationale to support a phase 1/2 clinical trial to test MSC exosomes in patients with osteochondral lesions.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Accelerate Diabetic Wound Healing via Ameliorating Oxidative Stress and Promoting Angiogenesis.

Diabetic wounds remain a great challenge for clinicians due to the multiple bacterial infections and oxidative damage. Exosomes, as an appealing nanodrug delivery system, have been widely applied in the treatment of diabetic wounds. Endovascular cells are important component cells of the vascular wall. Herein, we investigated the effects of HUCMSCs and HUC-Exos (exosomes secreted by HUCMSCs) on diabetic wound healing. In this study, HUVECs were coincubated with HUCMSCs, and HUC-Exos were utilized for in vitro and in vivo experiments to verify their roles in the regulation of diabetic wound healing. Our results demonstrated that HUCMSCs have the ability to regulate oxidative stress injuries of endothelial cells through exosomes and accelerate diabetic cutaneous wound healing in vitro. The present study suggests that HUC-Exos accelerate diabetic cutaneous wound healing, providing a promising therapeutic strategy for chronic diabetic wound repair.

Human bone marrow mesenchymal stem cells-derived exosomes attenuated prostate cancer progression via the miR-99b-5p/IGF1R axis

ABSTRACT MicroRNA-99b-5p (miR-99b-5p) has been shown to be enriched in serum exosomes of prostate cancer (PCa) patients treated with radiotherapy, while its function in PCa progression remains unclear. The expression levels of miR-99b-5p in PCa tissues, cancer cell lines and human bone marrow mesenchymal stem cells (HBMSCs), as well as HBMSCs-derived exosomes were assessed by quantitative real-time PCR (qRT-PCR). MiR-99b-5p mimics or inhibitor was transfected into HBMSCs, and HBMSCs-derived exosomes with abnormal expression of miR-99b-5p were used to stimulate PCa cell-line LNCaP cells. Cell proliferative rate was evaluated using Cell Counting Kit-8 (CCK-8) and 5‐ethynyl‐2′‐deoxyuridine (EdU) staining assays. Cell migration and invasion were analyzed by Transwell assay. The epithelial-mesenchymal transition (EMT) was evaluated by detecting EMT-related markers using Western blot analysis. The animal model was constructed to confirm the function of miR-99b-5p in vivo. The expression levels of MiR-99b-5p were decreased in PCa tissues and cell lines, while elevated in HBMSCs-derived exosomes. HBMSCs-derived exosomes significantly inhibited cell malignant phenotypes of PCa cells, and miR-99b-5p mimics transfected HBMSCs further enhanced the inhibitory effects of HBMSCs on PCa progression. In addition, miR-99b-5p inhibitor transfected HBMSCs-derived exosomes promoted the progression of PCa in vitro. Insulin-like growth factor 1 receptor (IGF1R) was identified as a downstream target of miR-99b-5p. Moreover, miR-99b-5p mimics transfected HBMSCs obviously inhibited tumor progression by downregulating IGF1R in animal model in vivo. Our results demonstrated that HBMSCs could attenuate PCa progression, and exosomal miR-99b-5p and IGF1R participated in the regulatory process, contributing to our understanding of the pathogenic mechanism of PCa.

Integrated Pipeline of Rapid Isolation and Analysis of Human Plasma Exosomes for Cancer Discrimination Based on Deep Learning of MALDI-TOF MS Fingerprints.

Plasma exosomes have shown great potential for liquid biopsy in clinical cancer diagnosis. Herein, we present an integrated strategy for isolating and analyzing exosomes from human plasma rapidly and then discriminating different cancers excellently based on deep learning fingerprints of plasma exosomes. Sequential size-exclusion chromatography (SSEC) was developed efficiently for separating exosomes from human plasma. SSEC isolated plasma exosomes, taking as less as 2 h for a single sample with high purity such that the discard rates of high-density lipoproteins and low/very low-density lipoproteins were 93 and 85%, respectively. Benefitting from the rapid and high-purity isolation, the contents encapsulated in exosomes, covered by plasma proteins, were well profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS). We further analyzed 220 clinical samples, including 79 breast cancer patients, 57 pancreatic cancer patients, and 84 healthy controls. After MS data pre-processing and feature selection, the extracted MS feature peaks were utilized as inputs for constructing a multi-classifier artificial neural network (denoted as Exo-ANN) model. The optimized model avoided overfitting and performed well in both training cohorts and test cohorts. For the samples in the independent test cohort, it realized a diagnosed accuracy of 80.0% with an area under the curve of 0.91 for the whole group. These results suggest that our integrated pipeline may become a generic tool for liquid biopsy based on the analysis of plasma exosomes in clinics.

Exosomes derived from adipose-derived stem cells alleviate cigarette smoke-induced lung inflammation and injury by inhibiting alveolar macrophages pyroptosis

BackgroundChronic obstructive pulmonary disease (COPD) is a frequently encountered disease condition in clinical practice mainly caused by cigarette smoke (CS). The aim of this study was to investigate the protective roles of human adipose-derived stem cells-derived exosomes (ADSCs-Exo) in CS-induced lung inflammation and injury and explore the underlying mechanism by discovering the effects of ADSCs-Exo on alveolar macrophages (AMs) pyroptosis.MethodsADSCs were isolated from human adipose tissues harvested from three healthy donors, and then ADSCs-Exo were isolated. In vivo, 24 age-matched male C57BL/6 mice were exposed to CS for 4 weeks, followed by intratracheal administration of ADSCs-Exo or phosphate buffered saline. In vitro, MH-S cells, derived from mouse AMs, were stimulated by 2% CS extract (CSE) for 24 h, followed by the treatment of ADSCs-Exo or phosphate buffered saline. Pulmonary inflammation was analyzed by detecting pro-inflammatory cells and mediators in the bronchoalveolar lavage fluid. Lung histology was assessed by hematoxylin and eosin staining. Mucus production was determined by Alcian blue-periodic acid-Schiff staining. The profile of AMs pyroptosis was evaluated by detecting the levels of pyroptosis-indicated proteins. The inflammatory response in AMs and the phagocytic activity of AMs were also investigated.ResultsIn mice exposed to CS, the levels of pro-inflammatory cells and mediators were significantly increased, mucus production was markedly increased and lung architecture was obviously disrupted. AMs pyroptosis was elevated and AMs phagocytosis was inhibited. However, the administration of ADSCs-Exo greatly reversed these alterations caused by CS exposure. Consistently, in MH-S cells with CSE-induced properties modelling those found in COPD, the cellular inflammatory response was elevated, the pyroptotic activity was upregulated while the phagocytosis was decreased. Nonetheless, these abnormalities were remarkably alleviated by the treatment of ADSCs-Exo.ConclusionsADSCs-Exo effectively attenuate CS-induced airway mucus overproduction, lung inflammation and injury by inhibiting AMs pyroptosis. Therefore, hADSCs-Exo may be a promising cell-free therapeutic candidate for CS-induced lung inflammation and injury.

Human bone marrow mesenchymal stem cells-derived exosomes protect against nerve injury via regulating immune microenvironment in neonatal hypoxic-ischemic brain damage model

Neonatal hypoxic-ischemic (HI) brain injury is a serious injury caused by various perinatal factors, which has become a heavy mental burden to the family. The molecular mechanism underlying neonatal hypoxic-ischemic brain injury remains largely unknown. Human bone marrow mesenchymal stem cells (hBMSCs) have caused wide public concern due to the immunomodulatory properties. Exosomes can polarize human microglia and thus changed it into an anti-inflammatory phenotype to reduce the release of pro-inflammatory factors. However, it is unclear whether hBMSCs-exosomes have effect on neonatal hypoxic-ischemic brain injury. In this study, we aimed at investigating the role of hBMSCs-exosomes in regulating immune response and nerve injury in neonatal hypoxic-ischemic brain damage model. In the research, we identified the exosome secretion of hBMSCs could transferred into human microglia (HMC). Moreover, we determined the importance of hBMSCs-exosomes in regulating HMC polarization and inflammatory response. Our research findings might provide a new insight into slowing the disease progression of neonatal hypoxic-ischemic brain injury.

Serum Exosomal mir-340-5p Promotes Angiogenesis in Brain Microvascular Endothelial Cells During Oxygen-Glucose Deprivation.

Ischemic stroke (IS) is a cerebrovascular disease with high morbidity, recurrence, and mortality. The purpose of the present study was to investigate the role and mechanism of human serum exosomes on angiogenesis after IS. The middle cerebral artery occlusion (MCAO) in vivo model and oxygen-glucose deprivation (OGD) in vitro model were established. Human serum exosomes from healthy samples (NC-exo) and IS samples (IS-exo) were injected into MCAO mice. Neurobehavioral tests were performed to assess the extent of neurological deficits. The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and the levels of inflammatory cytokines were analyzed by enzyme-linked immunosorbent assay (ELISA). In addition, human serum exosomes were cocultured with brain microvascular endothelial cells (BMECs). Cell Counting Kit-8 (CCK-8), Transwell, and tubule formation assays were performed to investigate the proliferation, migration, invasion, length, and branching of BMECs. The miRNA expression profiles of NC-exo and IS-exo were analyzed by high-throughput sequencing and compared. Bioinformatics and luciferase reporter assays were performed to evaluate the relationship between miR-340-5p and CD147. Serum NC-exo and IS-exo had protective effects on IS injury and promoted BMEC angiogenesis. Interestingly, the protective effect of IS-exo was weaker than that of NC-exo. In addition, miR-340-5p was downregulated in IS-exo, and miR-340-5p accelerated angiogenesis of BMECs after OGD. Mechanistically, CD147 was confirmed as a direct target of miR-340-5p. Finally, miR-340-5p promoted angiogenesis by directly targeting CD147. Serum exosome-derived miR-340-5p promote angiogenesis in OGD-induced BMECs by targeting CD147.

LncRNA and mRNA profiles of human milk-derived exosomes and their possible roles in protecting against necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is one of the most severe diseases commonly afflicting premature infants. Our previous studies suggests that human milk-derived exosomes (HM-Exos) have a potential therapeutic effect on NEC. In this study, we investigate the potentially therapeutic role of HM-Exos in an NEC animal model via comprehensive lncRNA and mRNA expression profiles. A rat model of NEC was induced through hypoxia, hypothermia and formula feeds. We extracted exosomes from the colostrum of healthy lactating mothers and identified their functions in an NEC animal model. Furthermore, high-throughput lncRNA and mRNA sequencings were explored to find the underlying mechanisms. Although both exosomes from term human breast milk (Term-Exos) and exosomes from preterm human breast milk (Pre-Exos) alleviated the severity of NEC, Pre-Exos seemed to better promote the proliferation of intestinal epithelial cells in vivo. We identified a total of 44 differentially expressed lncRNAs and 88 differentially expressed mRNAs between Term-Exos and Pre-Exos. Further GO and KEGG pathway analysis showed that the lncRNA-mRNA network of HM-Exos was associated with the JAK-STAT signaling pathway, bile secretion and the AMPK signaling pathway, which are predicted to be involved in the proliferation of cells. Therefore, this study reveals for the first time the important roles of human milk derived lncRNAs and mRNAs in protecting against necrotizing enterocolitis. These results provide new insight into the development of NEC.

Overexpression of lncRNA HCP5 in human umbilical cord mesenchymal stem cell-derived exosomes promoted the proliferation and inhibited the apoptosis of ovarian granulosa cells via the musashi RNA-binding protein 2/oestrogen receptor alpha 1 axis.

HCP5 has been reported to be downregulated in ovarian granulosa cells (OGCs) and to facilitate cell proliferation. Human umbilical cord mesenchymal stem cell exosome (hucMSCs-exo) treatment can prevent OGCs apoptosis in vitro. However, the functional mechanism of HCP5 and hucMSCs-exo requires further exploration. Fluorescence-activated cell sorting (FACS) was performed to measure the expression of markers related to hucMSCs. The osteogenic and adipogenic potential of hucMSCs was measured by alkaline phosphatase (ALP) and Alizarin red and by oil red-O staining, respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the mRNA and protein levels, respectively. Cell proliferation and apoptosis were measured by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and flow cytometry. The interaction of HCP5/musashi RNA-binding protein 2 (MSI2) and oestrogen receptor alpha 1 (ESR1) mRNA was analysed using RNA pulldown and RIP assays. HucMSCs and exosomes were successfully isolated and identified. HucMSC-derived exosomes promoted the proliferation of OGCs and ESR1 expression and inhibited cell apoptosis. HCP5 overexpression in exosomes further enhanced these effects. MSI2 knockdown led to the opposite results. HCP5 targeted MSI2, and MSI2 knockdown reduced the decreases in HCP5 and ESR1 expression. Mechanistically, HCP5 in HucMSC-derived exosomes promoted ESR1 expression by binding to MSI2, which promoted the proliferation of OGCs.

CircHIPK3 prevents cardiac senescence by acting as a scaffold to recruit ubiquitin ligase to degrade HuR.

Rational: Senescence is a major aging process that contributes to the development of cardiovascular diseases, but the underlying molecular mechanisms remain largely unknown. One reason is due to the lack of suitable animal models. We aimed to generate a cardiomyocyte (CM)-specific senescent animal model, uncover the underlying mechanisms, and develop new therapies for aging associated cardiac dysfunction. Methods: The gain/loss of circHIPK3 approach was used to explore the role of circHIPK3 in cardiomyocyte (CM) senescence. To investigate the mechanisms of circHIPK3 function in cardiac senescence, we generated CM-specific tamoxifen-induced circHIPK3 knockout (CKO) mice. We also applied various analyses including PCR, Western blot, nuclear and cytoplasmic protein extraction, immunofluorescence, echocardiography, RNA immunoprecipitation assay, RNA-pulldown assay, and co-immunoprecipitation. Results: Our novel CKO mice exhibited worse cardiac function, decreased circHIPK3 expression and telomere length shortening in the heart. The level of the senescence-inducer p21 in the hearts of CKO mice was significantly increased and survival was poor compared with control mice. In vitro, the level of p21 in CMs was significantly decreased by circHIPK3 overexpression, but increased by circHIPK3 silencing. We showed that circHIPK3 was a scaffold for p21 mRNA-binding protein HuR and E3 ubiquitin ligase β-TrCP. circHIPK3 silencing weakened the interaction between HuR and β-TrCP, reduced HuR ubiquitination, and enhanced the interaction between HuR and p21 mRNA. Moreover, we found that mice injected with human umbilical cord mesenchymal stem cell-derived exosomes (UMSC-Exos) showed increased circHIPK3 levels, decreased levels of p21, longer telomere length, and good cardiac function. However, these beneficial effects exerted by UMSC-Exos were inhibited by silencing circHIPK3. Conclusions: We successfully generated CM-specific CKO mice for aging research. Our results showed that deletion of circHIPK3 led to exaggerated CM senescence and decreased cardiac function. As a scaffold, circHIPK3 enhanced the binding of E3 ubiquitin ligase β-TrCP and HuR in the cytoplasm, leading to the ubiquitination and degradation of HuR and reduced p21 activity. In addition, UMSC-Exos exerted an anti-senescence and cardio-protective effect by delivering circHIPK3. These findings pave the way to the development of new therapies for aging associated cardiac dysfunction.

Global MicroRNA Profiling of Vascular Endothelial Cells.

MicroRNA sequencing (miRNA-seq) enables the detection and characterization of the cell miRNome, including miRNA isoforms (isomiRs) and novel miRNA species. In roughly half of the cases, the most abundant isomiR in the cells is not the reference miRNA given in miRBase, which highlights the importance of isomiR-specific analysis. Here, we describe a gel-free protocol for global miRNA profiling in vascular endothelial cells and the main steps of the subsequent data analysis with two alternative analysis methods. In addition to endothelial cells, the protocol is suitable for other cell and tissue types and has been successfully used to obtain miRNA-seq data from human cardiac tissue, plasma, pericardial fluid, and biofluid exosomes.

Protective effects of umbilical cord mesenchymal stem cell exosomes in a diabetic rat model through live retinal imaging.

To assess the protective effect of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exs) in a diabetic rat model by using a variety of retinal bioassays. hucMSCs were subjected to differential ultracentrifugation for the collection of exosomes, and transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) using a NanoSight analysis system and Western blotting (WB) were used to analyze the expression of surface marker proteins such as CD63, CD9 and Calnexin. Streptozotocin (STZ) was injected into the intraperitoneal cavity to establish a diabetic model. Rats were divided into a normal group, diabetic group and hucMSC-Ex group. Fundus fluorescein angiography (FFA), optical coherence tomography (OCT) and other live imaging methods were used to observe the fundus of the rats. Finally, the eyeballs of rats from each group were collected for hematoxylin-eosin (HE) staining to further analyze the retinal structure. Through TEM, NTA and WB, we successfully isolated hucMSC-Exs. Subsequent FFA and OCT confirmed that hucMSC-Exs effectively prevented early retinal vascular damage and thickening of the retina. Finally, HE staining of rat retinal sections revealed that exosomes effectively alleviated retinal structure disruption caused by diabetes. hucMSC-Exs have a protective effect on the retina in diabetic rat through FFA, OCT and HE staining.

Proteomic analysis and miRNA profiling of human testicular endothelial cell-derived exosomes: the potential effects on spermatogenesis.

Testicular endothelial cells have been found to play an important role in spermatogenesis and fertility, but their mechanism is obscure. Exosomes released by various cells are recognized as cell–cell communication mediators during the initiation and progression of many diseases. Therefore, the current study aimed to investigate the protein and miRNA components of human testicular endothelial cell-derived exosomes (HTEC-Exos) and to explore their potential effects on spermatogenesis. In this study, HTEC-Exos were first isolated by the ultracentrifugation method, and then identified by nanoparticle tracking analysis, transmission electron microscopy (TEM), and western blotting. The characteristics of HTEC-Exos were examined by liquid chromatography–mass spectrometry and microRNA (miRNA) chip analysis. Bioinformatics analysis was performed to explore the potential role of the exosomal content on spermatogenesis. A total of 945 proteins were identified, 11 of which were closely related to spermatogenesis. A total of 2578 miRNAs were identified. Among them, 30 miRNAs demonstrated potential associations with male reproductive disorders, such as azoospermia, and spermatogenesis disorders. In particular, 11 out of these 30 miRNAs have been proven to be involved in spermatogenesis based on available evidence. This study provides a global view of the proteins and miRNAs from HTEC-Exos, suggesting that HTEC-Exos may function as potential effectors during the process of spermatogenesis.

Human MSC-Derived Exosomes Reduce Cellular Senescence in Renal Epithelial Cells.

Cellular senescence of renal tubular cells is associated with chronic diseases and age-related kidney disorders. Therapies to antagonize senescence are, therefore, explored as novel approaches in nephropathy. Exosomes derived from human mesenchymal stroma-/stem-like cells (MSC) entail the transfer of multiple bioactive molecules, exhibiting profound regenerative potential in various tissues, including therapeutic effects in kidney diseases. Here, we first demonstrate that exosomes promote proliferation and reduce senescence in aged MSC cultures. For potential therapeutic perspectives in organ rejuvenation, we used MSC-derived exosomes to antagonize senescence in murine kidney primary tubular epithelial cells (PTEC). Exosome treatment efficiently reduced senescence while diminishing the transcription of senescence markers and senescence-associated secretory phenotype (SASP) factors. Concomitantly, we observed less DNA damage foci and more proliferating cells. These data provide new information regarding the therapeutic property of MSC exosomes in the development of renal senescence, suggesting a contribution to a new chapter of regenerative vehicles in senotherapy.

Serum-derived miR-574-5p-containing exosomes contribute to liver fibrosis by activating hepatic stellate cells

AimTo investigate the association of serum exosomes miR-574-5p with liver fibrosis, and explore the effect and mechanism of serum exosomes on HSC activation.Materials and methodsUsing serum samples collected from healthy adults and patients with liver cirrhosis, we extracted human serum exosomes via ultra-high-speed centrifugation, and co-cultured them with hepatic stellate cells (HSCs) line LX2. LX-2-mediated intake of human serum exosomes was examined by confocal microscopy. To induce liver fibrosis, we administered 20% CCl4 to mice intraperitoneally and adopted an exoEasy MIDI kit to extract serum exosomes.Liver fibrosis-related molecules were determined via qRT-PCR, Western blot, Masson staining, and Immunohistochemical staining.ResultsSignificantly high miR-574-5p levels were expressed in serum exosomes and were positively correlated with the expression of miR-574-5p, collagen deposition, and α-SMA expression in liver tissues of mice during liver fibrosis. Compared to healthy subjects, serum exosomes from cirrhosis patients were associated with higher expression of miR-574-5p. MiR-574-5p mimic promoted the expression of α-SMA and COL1A1 mRNA and protein in LX-2, whereas miR-574-5p inhibitor exerted no effect.ConclusionThis article demonstrates that miR-574-5p expression in serum exosomes is positively correlated with collagen deposition and HSC activation in liver tissues during liver fibrosis.Serum exosomes potentially activate HSC through the transfer of miR-574-5p to HSC during liver fibrosis.

Human amniotic fluid derived-exosomes alleviate hypoxic encephalopathy by enhancing angiogenesis in neonatal mice after hypoxia

Neonatal hypoxic encephalopathy is a type of central nervous system dysfunction manifested by high mortality and morbidity. Exosomes play a crucial role in neuroprotection by enhancing angiogenesis. The objective of this study was to investigate the effect of human amniotic fluid-derived exosomes (hAFEXOs) on functional recovery in neonatal hypoxic encephalopathy. The transwell assay, scratch wound healing assay, and tube formation assay were used to evaluate the effect of hAFEXOs on the angiogenesis of human umbilical vein endothelial cells (HUVECs) after oxygen and glucose deprivation (OGD). The angiogenesis of microvascular endothelial cells (MECs) in the cortex was tested in neonatal mice treated with hAFEXOs or phosphate-buffered saline (PBS) after hypoxia. Expressions of hypoxia-inducible factor 1 α (HIF-1α) and vascular endothelial growth factor (VEGF) in the cerebral cortex were also tested by western blot. The Morris Water Maze Test (MWM) was carried out to detect the performance of spatial memory after processing with hAFEXOs or PBS. The results indicated that hAFEXOs favored tubing formation and migration of HUVECs after in vitro OGD. The hAFEXOs also favored the expression of CD31 in neonatal mice following hypoxia. The expressions of both HIF-1α and VEGF were significantly augmented in the cerebral cortex of neonatal mice which were treated with hAFEXOs. Moreover, the MWM test results showed that the performance of the spatial memory was better in the hAFEXO-treated group than in the PBS-treated group. Our study indicates that hAFEXOs alleviated hypoxic encephalopathy and enhanced angiogenesis in neonatal mice after hypoxia. In addition, hAFEXOs promoted migration and tube formation of HUVECs after OGD in vitro. These findings confirm that hAFEXOs show great potential for further studies aimed at developing therapeutic agents for hypoxic encephalopathy.

Human umbilical cord mesenchymal stem cell-derived exosomes carrying hsa-miRNA-128-3p suppress pancreatic ductal cell carcinoma by inhibiting Galectin-3.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignant tumors of the digestive system. Many patients are diagnosed at an advanced stage and lose eligibility for surgery. Moreover, there are few effective methods for treating pancreatic ductal cell carcinoma. Increasing attention has been given to microRNAs (miRNAs) and their regulatory roles in tumor progression. In this study, we investigated the effects of exosomes extracted from human umbilical cord mesenchymal stem cells (HUCMSCs) carrying hsa-miRNA-128-3p on pancreatic cancer cells. Based on existing experimental and database information, we selected Galectin-3, which is associated with pancreatic cancer, and the corresponding upstream hsa-miRNA-128-3p. We extracted HUCMSCs from a fresh umbilical cord, hsa-miRNA-128-3p was transfected into HUCMSCs, and exosomes containing hsa-miRNA-128-3p were extracted and collected. The effect of exosomes rich in hsa-miRNA-128-3p on pancreatic cancer cells was analyzed. The expression of Galectin-3 in normal pancreatic duct epithelial cells was significantly lower than that in PDAC cell lines. We successfully extracted HUCMSCs from the umbilical cord and transfected hsa-miRNA-128-3p into HUCMSCs. Then we demonstrated that HUCMSC-derived exosomes with hsa-miRNA-128-3p could suppress the proliferation, invasion, and migration of PANC-1 cells in vitro by targeting Galectin-3. Hsa-miRNA-128-3p could be considered as a potential therapy for pancreatic cancer. We provided a new idea for targeted therapy of PDAC.

HPMSCs-Derived Exosomal miRNA-21 Protects Against Aging-Related Oxidative Damage of CD4+ T Cells by Targeting the PTEN/PI3K-Nrf2 Axis.

Mesenchymal stem cells (MSCs)-derived exosomes were considered a novel therapeutic approach in many aging-related diseases. This study aimed to clarify the protective effects of human placenta MSCs-derived exosomes (hPMSC-Exo) in aging-related CD4+ T cell senescence and identified the underlying mechanisms using a D-gal induced mouse aging model. Senescent T cells were detected SA-β-gal stain. The degree of DNA damage was evaluated by detecting the level of 8-OH-dG. The superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities were measured. The expression of aging-related proteins and senescence-associated secretory phenotype (SASP) were detected by Western blot and RT-PCR. We found that hPMSC-Exo treatment markedly decreased oxidative stress damage (ROS and 8-OH-dG), SA-β-gal positive cell number, aging-related protein expression (p53 and γ-H2AX), and SASP expression (IL-6 and OPN) in senescent CD4+ T cells. Additionally, hPMSC-Exo containing miR-21 effectively downregulated the expression of PTEN, increased p-PI3K and p-AKT expression, and Nrf2 nuclear translocation and the expression of downstream target genes (NQO1 and HO-1) in senescent CD4+ T cells. Furthermore, in vitro studies uncovered that hPMSC-Exo attenuated CD4+ T cell senescence by improving the PTEN/PI3K-Nrf2 axis by using the PTEN inhibitor bpV (HOpic). We also validated that PTEN was a target of miR-21 by using a luciferase reporter assay. Collectively, the obtained results suggested that hPMSC-Exo attenuates CD4+ T cells senescence via carrying miRNA-21 and activating PTEN/PI3K-Nrf2 axis mediated exogenous antioxidant defenses.