Mesenchymal Stem Cell Extracellular Vesicles Research Articles

Senescence of mesenchymal stem cells: implications in extracellular vesicles, miRNAs and their functional and therapeutic potentials

Senescence is seen as the cellular counterpart of tissue and biological aging, with irreversible stagnation of cell growth, and changes in function and behavior. Mesenchymal stem cells (MSCs) are one of the key therapeutic tools in regenerative medicine, and their regenerative and therapeutic potential declines significantly with the increasing age of cell donors and prolonged continuous culture in vitro. MicroRNAs (miRNAs) are regarded as important players in regulating the expression and function of multiple genes and pathways. Emerging evidence suggests that extracellular vesicles (EVs) participate in a complex cell senescence network, at least partially by providing certain miRNAs. Therefore, MSC EVs and miRNAs are implicated in not only contributing to but also influenced by MSC senescence. Here, we will provide an overview of the recent results on roles and mechanisms of miRNAs, particularly EV-miRNAs, involved in MSC senescence, and discuss their implications in functional properties and therapeutic efficacy of MSCs and their EVs. Keywords: Extracellular vesicles, microRNAs, mesenchymal stem cells, senescence

MicroRNA-based engineering of mesenchymal stem cell extracellular vesicles for treatment of retinal ischemic disorders: Engineered extracellular vesiclesand retinal ischemia.

Mesenchymal stem cell (MSCs)-derived extracellular vesicles (EVs) are emerging therapeutic tools. Hypoxic pre-conditioning (HPC) of MSCs altered the production of microRNAs (miRNAs) in EVs, and enhanced the cytoprotective, anti-inflammatory, and neuroprotective properties of their derivative EVs in retinal cells. EV miRNAs were identified as the primary contributors of these EV functions. Through miRNA seq analyses, miRNA-424 was identified as a candidate for the retina to overexpress in EVs for enhancing cytoprotection and anti-inflammatory effects. FEEs (functionally engineered EVs) overexpressing miR424 (FEE424) significantly enhanced neuroprotection and anti-inflammatory activities in vitro in retinal cells. FEE424 functioned by reducing inflammatory cytokine production in retinal microglia, and attenuating oxygen free radicals in retinal Muller cells and microvascular endothelial cells, providing a multi-pronged approach to enhancing recovery after retinal ischemic insult. In an in vivo model of retinal ischemia, native, HPC, and FEE424 MSC EVs robustly and similarly restored function to close to baseline, and prevented loss of retinal ganglion cells, but HPC EVs provided the most effective attenuation of apoptosis-related and inflammatory cytokine gene expression. These results indicate the potential for EV engineering to produce ameliorative effects for retinal diseases with a significant inflammatory component. STATEMENT OF SIGNIFICANCE: We show that functionally engineered extracellular vesicles (FEEs) from mesenchymal stem cells (MSCs) provide cytoprotection in rat retina subjected to ischemia. FEEs overexpressing microRNA 424 (FEE424) function by reducing inflammatory cytokine production in retinal microglia, and attenuating oxygen free radicals in Muller cells and microvascular endothelial cells, providing a multi-pronged approach to enhancing recovery. In an in vivo model of retinal ischemia in rats, native, hypoxic-preconditioned (HPC), and FEE424 MSC EVs robustly and similarly restored function, and prevented loss of retinal ganglion cells, but HPC EVs provided the most effective attenuation of apoptosis-related and inflammatory cytokine gene expression. The results indicate the potential for EV engineering to produce ameliorative effects for retinal diseases with a significant inflammatory component.

Micro RNA based MSC EV engineering: Targeting the BMP2 cascade for bone repair.

Mesenchymal stem cell derived extracellular vesicles (MSC EVs) possess excellent immunomodulatory and therapeutic properties. While beneficial, from a translational perspective, extracellular vesicles with consistent functionality and target specificity are required to achieve the goals of precision medicine and tissue engineering. Prior research has identified that the miRNA composition of mesenchymal stem cell derived extracellular vesicles contributes significantly towards extracellular vesicles functionality. In this study, we hypothesized that mesenchymal stem cell derived extracellular vesicle functionality can be rendered pathway-specific using a miRNA-based extracellular vesicles engineering approach. To test this hypothesis, we utilized bone repair as a model system and the BMP2 signaling cascade as the targeted pathway. We engineered mesenchymal stem cell extracellular vesicles to possess increased levels of miR-424, a potentiator of the BMP2 signaling cascade. We evaluated the physical and functional characteristics of these extracellular vesicles and their enhanced ability to trigger the osteogenic differentiation of naïve mesenchymal stem cell in vitro and facilitate bone repair in vivo. Results indicated that the engineered extracellular vesicles retained their extracellular vesicles characteristics and endocytic functionality and demonstrated enhanced osteoinductive function by activating SMAD1/5/8 phosphorylation and mesenchymal stem cell differentiation in vitro and enhanced bone repair in vivo. Furthermore, the inherent immunomodulatory properties of the mesenchymal stem cell derived extracellular vesicles remained unaltered. These results serve as a proof-of-concept for miRNA-based extracellular vesicles engineering approaches for regenerative medicine applications.

Second International Pulmonary Hypertension/Heart Failure Symposium—Structural heart disease, right ventricular dysfunction, and stem cell therapy: The European Pediatric Pulmonary Vascular Disease Network

Second International Pulmonary Hypertension/Heart Failure Symposium—Structural heart disease, right ventricular dysfunction, and stem cell therapy: The European Pediatric Pulmonary Vascular Disease Network

The Emerging Role of Extracellular Vesicles from Mesenchymal Stem Cells and Macrophages in Pulmonary Fibrosis: Insights into miRNA Delivery.

Pulmonary fibrosis is a type of chronic, progressive, fibrotic lung disease of unclear cause with few treatment options. Cell therapy is emerging as a promising novel modality for facilitating lung repair. Mesenchymal stem cell (MSC)-based and macrophage-based cell therapies are regarded as promising strategies to promote lung repair, due to incredible regenerative potential and typical immunomodulatory function, respectively. Extracellular vesicles (EVs), including exosomes and microvesicles, are cell-derived lipid-bilayer membrane vesicles that are secreted from virtually every cell and are involved in intercellular communication by delivering expansive biological cargos to recipients. This review provides a deep insight into the recent research progress concerning the effects of MSC and macrophage-associated EVs on the pathogenesis of pulmonary fibrosis. In addition to discussing their respective vital roles, we summarize the importance of cross-talk, as macrophages are vital for MSCs to exert their protective effects through two major patterns, including attenuating macrophage activation and M1 phenotype macrophage polarization. Moreover, miRNAs are selectively enriched into EVs as essential components, and consideration is given to the particular effects of EV-associated miRNAs.

Mesenchymal Stem Cell-Derived Extracellular Vesicles as Idiopathic Pulmonary Fibrosis Microenvironment Targeted Delivery.

Idiopathic pulmonary fibrosis (IPF) affects an increasing number of people globally, yet treatment options remain limited. At present, conventional treatments depending on drug therapy do not show an ideal effect in reversing the lung damage or extending the lives of IPF patients. In recent years, more and more attention has focused on extracellular vesicles (EVs) which show extraordinary therapeutic effects in inflammation, fibrosis disease, and tissue damage repair in many kinds of disease therapy. More importantly, EVs can be modified or used as a drug or cytokine delivery tool, targeting injury sites to enhance treatment efficiency. In light of this, the treatment strategy of mesenchymal stem cell-extracellular vesicles (MSC-EVs) targeting the pulmonary microenvironment for IPF provides a new idea for the treatment of IPF. In this review, we summarized the inflammation, immune dysregulation, and extracellular matrix microenvironment (ECM) disorders in the IPF microenvironment in order to reveal the treatment strategy of MSC-EVs targeting the pulmonary microenvironment for IPF.

A new cell-free therapeutic strategy for liver regeneration: Human placental mesenchymal stem cell-derived extracellular vesicles

Mesenchymal stem cells (MSCs) have potential role in organ regeneration therapy. Previous work indicating that MSCs confer protection against liver disease. Here, we aimed to determine the potential application in liver regeneration of human placenta-derived MSCs extracellular vesicles (hPMSCs-EVs) via experimental hepatectomy. hPMSCs-EVs were administered intravenously 24 h before 70% partial hepatectomy, the specific composition of hPMSCs-EVs was identified by sequencing and validated by the quantitative polymerase chain reaction, including circ-RBM23. The role of circ-RBM23 in L02 cell was evaluated and it was found that circ-RBM23 knockdown inhibited L02 cell proliferation both in vitro and in vivo. The competing endogenous RNA function of circ-RBM23 was evaluated by the RNA immunoprecipitation assay and found that circ-RBM23 shares miRNA response elements with RRM2. Overexpressed circ-RBM23 bound competitively to miR-139-5p, preventing the miRNA-mediated degradation of RRM2, activating the expression of eIF4G and AKT/mTOR, and facilitating liver regeneration. These results indicate that hPMSCs-EVs prevent hepatic dysfunction and improve liver regeneration in vivo and hepatocytes proliferation in vitro, potentially via circ-RBM23 delivery.

Mesenchymal stem cell extracellular vesicles mitigate vascular permeability and injury in the small intestine and lung in a mouse model of hemorrhagic shock and trauma.

Hemorrhagic shock and trauma (HS/T)-induced gut injury may play a critical role in the development of multi-organ failure. Novel therapies that target gut injury and vascular permeability early after HS/T could have substantial impacts on trauma patients. In this study, we investigate the therapeutic potential of human mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC EVs) in vivo in HS/T in mice and in vitro in Caco-2 human intestinal epithelial cells. In vivo, using a mouse model of HS/T, vascular permeability to a 10-kDa dextran dye and histopathologic injury in the small intestine and lungs were measured among mice. Groups were (1) sham, (2) HS/T + lactated Ringer's (LR), (3) HS/T + MSCs, and (4) HS/T + MSC EVs. In vitro, Caco-2 cell monolayer integrity was evaluated by an epithelial cell impedance assay. Caco-2 cells were pretreated with control media, MSC conditioned media (CM), or MSC EVs, then challenged with hydrogen peroxide (H2O2). In vivo, both MSCs and MSC EVs significantly reduced vascular permeability in the small intestine (fluorescence units: sham, 456 ± 88; LR, 1067 ± 295; MSC, 765 ± 258; MSC EV, 715 ± 200) and lung (sham, 297 ± 155; LR, 791 ± 331; MSC, 331 ± 172; MSC EV, 303 ± 88). Histopathologic injury in the small intestine and lung was also attenuated by MSCs and MSC EVs. In vitro, MSC CM but not MSC EVs attenuated the increased permeability among Caco-2 cell monolayers challenged with H2O2. Mesenchymal stem cell EVs recapitulate the effects of MSCs in reducing vascular permeability and injury in the small intestine and lungs in vivo, suggesting MSC EVs may be a potential cell-free therapy targeting multi-organ dysfunction in HS/T. This is the first study to demonstrate that MSC EVs improve both gut and lung injury in an animal model of HS/T.

Mesenchymal Stem Cell Derived Extracellular Vesicles Reverse Radiation-Induced Cytokine Storm

Cytokine storm or cytokine release syndrome is a systemic inflammatory response to different triggers which leads to excessive activation of immune cells with the release of a large amount of pro-inflammatory cytokines. These uncontrolled and excessive releases of cytokines accompany multisystem organ failure and death. Acute radiation exposure leads to acute hematopoietic and acute gastrointestinal syndromes with early mortality. Cytokine storm accompanies these syndromes and significantly complicates the clinical outcomes.Recent data has suggested that the inflammasome and associate cytokine storm may play a major role in various injuries and tissue problems after irradiation. Inflammasome activates caspase-1 which give rise to the proinflammatory cytokines such as interleukin-18 (IL-18) and interleukin-1 beta (IL-1β) into their biologically active forms and further induce other cytokines release.Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles, released from cells in the whole body. EVs play an important role in intercellular communication. EVs have been shown to transfer protein, lipid, DNA, mRNAs, and microRNAs to recipient cells, thereby mediating a variety of biological responses. We have been investigating the capacity of marrow-derived mesenchymal stem cell extracellular vesicles (MSC-EV) to reverse radiation damage to murine bone marrow stem/progenitor cells.In this study, we have evaluated the effect of MSC-EVs on reversal of high dose radiation injury to bone marrow cells. C57BL/6 mice received 0, 700, and 950-1000cGy WBI (either single or split dose). After 24 hrs. post-irradiation, mice received an IV infusion of 2X10 9 hMSC EVs daily for three days with a control group receiving vehicle only. Marrow was harvested from 700 cGy exposed mice which were vesicle treated or not. These marrow cells were evaluated for engraftment into 950 cGy exposed mice. The engraftment rates in mice transplanted with marrow from irradiated EV injected animals was 34.66±14.19% at 4 months post transplantation and marrow from the group not treated with EV gave an engraftment rate of 6.92±3.53%. We further evaluated the effect of MSCs-EVs on decreasing the mortality in mice exposed to 950cGy WBI. The MSC-EV untreated mice were dead between 12-18 days post radiation, but MSC-EV treated mice maintained a 60% survival rate at 130 days post radiation, suggesting that MSC-EV treatment could significantly extend the survival rate of mice after exposure to lethal radiation. We evaluated the circulating inflammatory markers in 1000 cGy whole body irradiated mice with/without MSC-EV treatment. Twenty-four hours post-irradiation, mice received an IV infusion of 2x10 9 hMSC EVs every day for three days with a control group receiving vehicle only. The serum was collected after 8 days post radiation for inflammatory response analysis by ProcartaPlex multiplex immunoassays. There were dramatic increases in cytokines secretion in serum including IFN gamma, IL-12p70, IL-13, IL-1 beta, IL-2, IL-5, IL-6, TNF alpha, GM-CSF and IL-18 in irradiated mice compared to non-irradiated mice and a significant reduction in cytokine levels after MSC-EVs treatment. We further found significantly higher levels of IL-1 beta and activated caspase-1 p20 protein expression in the spleen after 1000cGy radiation compared to non-radiated mice. Moreover, we found that with MSC-EV treatment, the expression level of IL-1 beta and active caspase-1 p20 was significantly inhibited in spleen compared to untreated irradiated mice, indicating that the inhibition of cytokine storm post-radiation by MSC-EVs might be mediated by inhibition of inflammasome activation. Our study suggests that EV Inhibition of an inflammasome mediated cytokine storm could be therapeutically important in many disease entities. DisclosuresNo relevant conflicts of interest to declare.

Effect of dose, dosing intervals, and hypoxic stress on the reversal of pulmonary hypertension by mesenchymal stem cell extracellular vesicles.

RationaleMesenchymal stem cell extracellular vesicles (MSC EVs) reverse pulmonary hypertension, but little information is available regarding what dose is effective and how often it needs to be given. This study examined the effects of dose reduction and use of longer dosing intervals and the effect of hypoxic stress of MSC prior to EV collection.MethodsAdult male rats with pulmonary hypertension induced by Sugen 5416 and three weeks of hypoxia (SuHx-pulmonary hypertension) were injected with MSC EV or phosphate buffered saline the day of removal from hypoxia using one of the following protocols: (1) Once daily for three days at doses of 0.2, 1, 5, 20, and 100 µg/kg, (2) Once weekly (100 µg/kg) for five weeks, (3) Once every other week (100 µg/kg) for 10 weeks, (4) Once daily (20 µg/kg) for three days using EV obtained from MSC exposed to 48 h of hypoxia (HxEV) or MSC kept in normoxic conditions (NxEV).Main resultsMSC EV reversed increases in right ventricular systolic pressure (RVSP), right ventricular to left ventricle + septum weight (RV/LV+S), and muscularization index of pulmonary vessels ≤50 µm when given at doses of 20 or 100 μg/kg. RVSP, RV/LV+S, and muscularization index were significantly higher in SuHx-pulmonary hypertension rats treated once weekly with phosphate buffered saline for five weeks or every other week for 10 weeks than in normoxic controls, but not significantly increased in SuHx-pulmonary hypertension rats given MSC EV. Both NxEV and HxEV significantly reduced RVSP, RV/LV+S, and muscularization index, but no differences were seen between treatment groups.ConclusionsMSC EV are effective at reversing SuHx-pulmonary hypertension when given at lower doses and longer dosing intervals than previously reported. Hypoxic stress does not enhance the efficacy of MSC EV at reversing pulmonary hypertension. These findings support the feasibility of MSC EV as a long-term treatment for pulmonary hypertension.

Mesenchymal Stem Cell Extracellular Vesicles as Adjuvant to Bone Marrow Stimulation in Chondral Defect Repair in a Minipig Model.

This study evaluated the effects of mesenchymal stem cell-extracellular vesicles (MSC-EVs) on chondrocyte proliferation in vitro and on cartilage repair in vivo following bone marrow stimulation (BMS) of focal chondral defects of the knee. Six adult Göttingen minipigs received 2 chondral defects in each knee. The pigs were randomized to treatment with either BMS combined with MSC-EVs or BMS combined with phosphate-buffered saline (PBS). Intraarticular injections MSC-EVs or PBS were performed immediately after closure of the surgical incisions, and at 2 and 4 weeks postoperatively. Repair was evaluated after 6 months with gross examination, histology, histomorphometry, immunohistochemistry, and micro-computed tomography (µCT) analysis of the trabecular bone beneath the defect. Defects treated with MSC-EVs had more bone in the cartilage defect area than the PBS-treated defects (7.9% vs. 1.5%, P = 0.02). Less than 1% of the repair tissue in both groups was hyaline cartilage. International Cartilage and Joint Preservation Society II histological scoring showed that defects treated with MSC-EVs scored lower on "matrix staining" (20.8 vs. 50.0, P = 0.03), "cell morphology" (35.4 vs. 53.8, P = 0.04), and "overall assessment" (30.8 vs. 52.9, P = 0.03). Consistently, defects treated with MSC-EVs had lower collagen II and higher collagen I areal deposition. Defects treated with MSC-EVs had subchondral bone with significantly higher tissue mineral densities than PBS-treated defects (860 mg HA/cm3 vs. 838 mg HA/cm3, P = 0.02). Intraarticular injections of MSC-EVs in conjunction with BMS led to osseous ingrowth that impaired optimal cartilage repair, while enhancing subchondral bone healing.

Mesenchymal stem cell-derived extracellular vesicles as probable triggers of radiation-induced heart disease

BackgroundRadiation-induced heart disease has been reported, but the underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs), also residing in the heart, are highly susceptible to radiation. We examined the hypothesis that the altered secretion of extracellular vesicles (EVs) from MSCs is the trigger of radiation-induced heart disease.MethodsBy exposing human placental tissue-derived MSCs to 5 Gy γ-rays, we then isolated EVs from the culture medium 48 h later and evaluated the changes in quantity and quality of EVs from MSCs after radiation exposure. The biological effects of EVs from irradiated MSCs on HUVECs and H9c2 cells were also examined.ResultsAlthough the amount and size distribution of EVs did not differ between the nonirradiated and irradiated MSCs, miRNA sequences indicated many upregulated or downregulated miRNAs in irradiated MSCs EVs. In vitro experiments using HUVEC and H9c2 cells showed that irradiated MSC-EVs decreased cell proliferation (P < 0.01), but increased cell apoptosis and DNA damage. Moreover, irradiated MSC-EVs impaired the HUVEC tube formation and induced calcium overload in H9c2 cells.ConclusionsEVs released from irradiated MSCs show altered miRNA profiles and harmful effects on heart cells, which provides new insight into the mechanism of radiation-related heart disease risks.

Treating amyotrophic lateral sclerosis with a bone marrow derived mesenchymal stem cell extracellular vesicles - A case report

Amyotrophic Lateral Sclerosis (ALS) is a terminal neurodegenerative disease with only a few minimally effective treatment options. This study treats a patient with Spinal ALS with Bone Marrow Derived Mesenchymal Stem Cell Extracellular Vesicles (BM-MSCEVs) to slow or stop the progress of the disease. After 2 months and 4 treatments, the patient demonstrated improvement in ALS functional scores and most physical exam findings. BM-MSCEVs demonstrate the ability to effectively treat Spinal ALS in this study and open the door for future studies.

Reversal of Aplastic Anemia By Mesenchymal Stem Cell-Derived Extracellular Vesicles

Aplastic anemia is a disease of bone marrow failure. Aplastic anemia remains a challenging disease as far as etiology and therapeutic approaches. Extracellular vesicles (EVs) have been studied for many years. These were initially felt to be cell junk largely from platelets and erythrocytes. But, more recently, intense interest has been focused on their potential for altering cell fate and reversing tissue injury. Vesicles derived from mesenchymal stem cells (MSC) have been shown to affect the phenotype and induce healing of many different cell types. Our recent published work has shown that human or murine MSC-EV treatment dramatically reversed radiation damage to bone marrow both in vitro and in vivo by inhibition of DNA damage and apoptosis. In this study, we evaluated whether human MSC-EVs could reverse marrow failure in a Busulfan induced aplastic anemia mouse model. We have carried out studies on Busulfan (BU) induced marrow aplasia by administering Busulfan in doses of 9.25 mg/kg intraperitoneally (9 times over 20 days) to induce bone marrow aplasia in female Balb/cByJ mice. Mice were then infused with 4x108 human MSC-EVs at 1, 2 and 3 days after the final BU dose. We observed that the human MSC EV treatment significantly prolonged survival in these mice. 50% of the BU treated mice died by 30 days after BU injection, whereas only 30% of EV-injected mice died by this time. The mice were sacrificed to harvest WBM cells and peripheral blood for cell count at 10 days after the final BU dose. There were significant decreases in marrow cellularity from tibiae and femurs and complete blood count (CBC) including RBC, WBC, HBG and PLT in BU treated mice compared to vehicle control mice. The vesicle treatment led to a statistically significant (p<0.05) increase in bone marrow cell count with the average of 3.30 x106 ±0.46 x106 cells /tibia and femur for the vesicle treated mice compared to 2.05 x106 ± 0.46 x 106 cells for the non-vesicle treated mice. The restoration growth of bone marrow stem cells was further confirmed by WBM proliferation assessment in cell culture. We further investigated the effect of MSC-EVs on the microRNA expression in bone marrow cells from Busulfan induced aplastic anemia mice by microRNA realtime PCR array. 151 microRNAs were altered by Busulfan treatment. 88 showed partial or complete reversal of these alterations after EV treatment. In summary, our data suggest that MSC-EV treatment could partially reverse Busulfan induced marrow failure at early time points. The reversal of the Busulfan induced chronic bone marrow failure by MSC-EVs needs further investigation. DisclosuresNo relevant conflicts of interest to declare.

Proteomic analysis of bone marrow-derived mesenchymal stem cell extracellular vesicles from healthy donors: implications for proliferation, angiogenesis, Wnt signaling, and the basement membrane

BackgroundBone marrow-derived mesenchymal stem cells (BM-MSCs) have shown therapeutic potential in various in vitro and in vivo studies in cutaneous wound healing. Furthermore, there are ubiquitous studies highlighting the pro-regenerative effects of BM-MSC extracellular vesicles (BM-MSC EVs). The similarities and differences in BM-MSC EV cargo among potential healthy donors are not well understood. Variation in EV protein cargo is important to understand, as it may be useful in identifying potential therapeutic applications in clinical trials. We hypothesized that the donors would share both important similarities and differences in cargo relating to cell proliferation, angiogenesis, Wnt signaling, and basement membrane formation—processes shown to be critical for effective cutaneous wound healing.MethodsWe harvested BM-MSC EVs from four healthy human donors who underwent strict screening for whole bone marrow donation and further Good Manufacturing Practices-grade cell culture expansion for candidate usage in clinical trials. BM-MSC EV protein cargo was determined via mass spectrometry and Proteome Discoverer software. Corresponding proteomic networks were analyzed via the UniProt Consortium and STRING consortium databases.ResultsMore than 3000 proteins were identified in each of the donors, sharing > 600 proteins among all donors. Despite inter-donor variation in protein identities, there were striking similarities in numbers of proteins per biological functional category. In terms of biologic function, the proteins were most associated with transport of ions and proteins, transcription, and the cell cycle, relating to cell proliferation. The donors shared essential cargo relating to angiogenesis, Wnt signaling, and basement membrane formation—essential processes in modulating cutaneous wound repair.ConclusionsHealthy donors of BM-MSC EVs contain important similarities and differences among protein cargo that may play important roles in their pro-regenerative functions. Further studies are needed to correlate proteomic signatures to functional outcomes in cutaneous repair.

Role of CD44 in increasing the potency of mesenchymal stem cell extracellular vesicles by hyaluronic acid in severe pneumonia

BackgroundAlthough promising, clinical translation of human mesenchymal stem or stromal cell-derived extracellular vesicles (MSC EV) for acute lung injury is potentially limited by significant production costs. The current study was performed to determine whether pretreatment of MSC EV with high molecular weight hyaluronic acid (HMW HA) would increase the therapeutic potency of MSC EV in severe bacterial pneumonia.MethodsIn vitro experiments were performed to determine the binding affinity of HMW HA to MSC EV and its uptake by human monocytes, and whether HMW HA primed MSC EV would increase bacterial phagocytosis by the monocytes. In addition, the role of CD44 receptor on MSC EV in the therapeutic effects of HMW HA primed MSC EV were investigated. In Pseudomonas aeruginosa (PA) pneumonia in mice, MSC EV primed with or without HMW HA were instilled intravenously 4 h after injury. After 24 h, the bronchoalveolar lavage fluid, blood, and lungs were analyzed for levels of bacteria, inflammation, MSC EV trafficking, and lung pathology.ResultsMSC EV bound preferentially to HMW HA at a molecular weight of 1.0 MDa compared with HA with a molecular weight of 40 KDa or 1.5 MDa. HMW HA primed MSC EV further increased MSC EV uptake and bacterial phagocytosis by monocytes compared to treatment with MSC EV alone. In PA pneumonia in mice, instillation of HMW HA primed MSC EV further reduced inflammation and decreased the bacterial load by enhancing the trafficking of MSC EV to the injured alveolus. CD44 siRNA pretreatment of MSC EV prior to incubation with HMW HA eliminated its trafficking to the alveolus and therapeutic effects.ConclusionsHMW HA primed MSC EV significantly increased the potency of MSC EV in PA pneumonia in part by enhancing the trafficking of MSC EV to the sites of inflammation via the CD44 receptor on MSC EV which was associated with increased antimicrobial activity.

Surface engineering of titania nanotubes incorporated with double-layered extracellular vesicles to modulate inflammation and osteogenesis.

Titania nanotubes (TNT) generated on titanium implant are emerged as important modification technique to facilitate bone regeneration. Mesenchymal stem cells (MSCs)-derived exosomes are membrane bound extracellular vesicles (EVs), which play an important role in tissue regeneration. The objective of this study was to generate an EVs hybrid TNT aiming at regulating inflammation, MSCs recruitment and osteogenesis. We isolated EVs from MSCs (MSCs EVs) and 3-day osteogenically differentiated MSCs (3d EVs). MSC EVs and 3d EVs exhibited round morphology under TEM, which also showed robust internalization by human bone marrow derived MSCs (hBMSCs). Next, we fabricated 3d EVs/MSC EVs hybrid TNT. When inflammatory macrophages were co-cultured with EVs hybrid TNT, the gene and protein expression of inflammatory cytokine were significantly reduced. Macrophage morphology was also examined by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Further migratory ability study using hBMSCs indicated significant enhancement of MSCs migration in EVs hybrid TNT. In addition, we further demonstrated significant increase of osteogenic differentiation of hBMSCs in EVs hybrid TNT. This study suggests that EVs hybrid TNT may serve as a viable therapeutic approach to enhance osteogenesis and bone regeneration.

Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

3D Encapsulation and tethering of functionally engineered extracellular vesicles to hydrogels

Mesenchymal stem cell (MSC) derived extracellular vesicles (EVs) in their naïve and engineered forms have emerged as potential alternatives to stem cell therapy. While they have a defined therapeutic potential, the spatial and temporal control of their activity in vivo remains a challenge. The objective of this study was to devise a methodology to encapsulate EVs in 3D hydrogels for prolonged delivery. To achieve this, we have leveraged the MSC EV interactions with ECM proteins and their derivative peptides. Using osteoinductive functionally engineered EVs (FEEs) derived from MSCs, we show that FEEs bind to mimetic peptides from collagen (DGEA, GFPGER) and fibronectin (RGD). In in vitro experiments, photocrosslinkable alginate hydrogels containing RGD were able to encapsulate, tether and retain the FEEs over a period of 7 days while maintaining the structural integrity and osteoinductive functionality of the EVs. When employed in a calvarial defect model in vivo, alginate-RGD hydrogels containing the FEEs enhanced bone regeneration by a factor of 4 compared to controls lacking FEEs and by a factor of 2 compared to controls lacking the tethering peptide. These results show that EVs can be tethered to biomaterials to promote bone repair and the importance of prolonged delivery in vivo. Results also provide a prelude to the possible use of this technology for controlled delivery of EVs for other regenerative medicine applications. Statement of significanceThe beneficial effects of human MSC (HMSC) therapy are attributable to paracrine effects of the HMSC derived EVs. While EV engineering has the potential to impact several fields of regenerative medicine, targeted delivery of the engineered EVs with spatial and temporal control is necessary to prevent off-target effects and enhance tissue specificity. Here, we have leveraged the interactions of MSC EVs with ECM proteins to develop a tethering system that can be utilized to prolong EV delivery in vivo while maintaining the structural and functional integrity of the EVs. Our work has provided a tunable platform for EV delivery that we envision can be formulated as an injectable material or a bulk hydrogel suitable for regenerative medicine applications.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Regenerative Potential and Challenges.

Simple SummaryMesenchymal stem cell extracellular vesicles (MSCEVs) obtained from MSCs can have numerous therapeutic applications via regeneration of various body tissues. There are certain approaches by which the therapeutic effect of MSCEVs can be further potentiated. Translation of MSCEVs from the preclinical to clinical level presents several challenges to investigators. Thus, knowledge of isolation, culturing, application, and various challenges faced during clinical applications of MSCEVs are the important aspects highlighted in the present review.Evidence suggests that stem cells exert regenerative potential via the release of extracellular vesicles. Mesenchymal stem cell extracellular vesicles (MSCEVs) offer therapeutic benefits for various pathophysiological ailments by restoring tissues. Facts suggest that MSCEV action can be potentiated by modifying the mesenchymal stem cells culturing methodology and bioengineering EVs. Limited clinical trials of MSCEVs have questioned their superiority, culturing quality, production scale-up and isolation, and administration format. Translation of preclinically successful MSCEVs into a clinical platform requires paying attention to several critical matters, such as the production technique, quantification/characterization, pharmacokinetics/targeting/transfer to the target site, and the safety profile. Keeping these issues as a priority, the present review was designed to highlight the challenges in translating preclinical MSCEV research into clinical platforms and provide evidence for the regenerative potential of MSCEVs in various conditions of the liver, kidney, heart, nervous system, bone, muscle, cartilage, and other organs/tissues.