The Role of Extracellular Vesicles in the Progression of Human Neuroblastoma

The long-underestimated role of extracellular vesicles in cancer is now reconsidered worldwide by basic and clinical scientists, who recently highlighted novel and crucial activities of these moieties. Extracellular vesicles are now considered as king transporters of specific cargoes, including molecular components of parent cells, thus mediating a wide variety of cellular activities both in normal and neoplastic tissues. Here, we discuss the multifunctional activities and underlying mechanisms of extracellular vesicles in neuroblastoma, the most frequent common extra-cranial tumor in childhood. The ability of extracellular vesicles to cross-talk with different cells in the tumor microenvironment and to modulate an anti-tumor immune response, tumorigenesis, tumor growth, metastasis and drug resistance will be pinpointed in detail. The results obtained on the role of extracellular vesicles may represent a panel of suggestions potentially useful in practice, due to their involvement in the response to chemotherapy, and, moreover, their ability to predict resistance to standard therapies—all issues of clinical relevance.     

Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration

SmartBone^® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.     

Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA,VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.     

Advances and Perspectives in Dental Pulp Stem Cell Based Neuroregeneration Therapies

Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types for regenerative therapies given their ability to grow in the absence of serum and their realistic possibility to be used in autologous grafts. In this review, we describe the particular advantages of hDPSCs for neuroregenerative cell therapies. We thoroughly discuss the knowledge about their embryonic origin and characteristics of their postnatal niche, as well as the current status of cell culture protocols to maximize their multilineage differentiation potential, highlighting some common issues when assessing neuronal differentiation fates of hDPSCs. We also review the recent progress on neuroprotective and immunomodulatory capacity of hDPSCs and their secreted extracellular vesicles, as well as their combination with scaffold materials to improve their functional integration on the injured central nervous system (CNS) and peripheral nervous system (PNS). Finally, we offer some perspectives on the current and possible future applications of hDPSCs in neuroregenerative cell therapies.     

Extracellular Vesicles as a Therapeutic Tool for Kidney Disease: Current Advances and Perspectives

Extracellular vesicles (EVs) have been described as important mediators of cell communication, regulating several physiological processes, including tissue recovery and regeneration. In the kidneys, EVs derived from stem cells have been shown to support tissue recovery in diverse disease models and have been considered an interesting alternative to cell therapy. For this purpose, however, several challenges remain to be overcome, such as the requirement of a high number of EVs for human therapy and the need for optimization of techniques for their isolation and characterization. Moreover, the kidney’s complexity and the pathological process to be treated require that EVs present a heterogeneous group of molecules to be delivered. In this review, we discuss the recent advances in the use of EVs as a therapeutic tool for kidney diseases. Moreover, we give an overview of the new technologies applied to improve EVs’ efficacy, such as novel methods of EV production and isolation by means of bioreactors and microfluidics, bioengineering the EV content and the use of alternative cell sources, including kidney organoids, to support their transfer to clinical applications.     

Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA⁺CD41⁺ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA⁺ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.     

Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk

Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.     

Renoprotective Angiographic Polymersomes

Computed tomography (CT) angiography is powerful for the diagnosis of vascular diseases. Unfortunately, this method usually requires a high dosage of iodinated contrast agents, which can lead to severe elevation of reactive oxygen species (ROS) levels within kidneys. This causes oxidative stress, apoptosis, and hence contrast-induced nephropathy (CIN), a leading cause of iatrogenic renal failure, especially for patients with renal insufficiency. Herein, a route is shown to circumvent such problems with the usage of rationally designed renoprotective angiographic polymersomes (RAPs) as blood pool CT contrast agents. RAPs are biodegradable nanoparticles prepared via self-assembly of poly(ethylene oxide)-block-poly(triiodobenzoic chloride-conjugated polylysine-stat-phenylboronic acid pinacol ester-conjugated polylysine) (PEO₄₅-b-P[(Lys-IBC)₄₅-stat-(Lys-PAPE)₁₅]). The key to the efficiency of such nanoparticles as renoprotective contrast agents arises from the rationally chosen repeat units: Lys-IBC exhibits a concentration-dependent X-ray attenuation capability and Lys-PAPE introduces an ROS-scavenging ability to the polymersome. The study shows that RAPs can reduce the risk of CIN in mice with kidney injury. Additionally, a 5-fold increase in angiographic live time is observed using RAPs, compared to commonly used iodinated small molecule contrast agents. In summary, a new strategy is proposed for the design of a renoprotective angiographic contrast agent that is capable of reducing the risk of CIN.     

Surfactant-Assisted Purification of Hydrophobic DNA Nanostructures

Purification of functional DNA nanostructures is an essential step in achieving intended functions because misfolded structures and the remaining free DNA strands in a solution can interact and affect their behavior. However, due to hydrophobicity-mediated aggregation, it is difficult to purify DNA nanostructures modified with hydrophobic molecules by conventional methods. Herein, we report the purification of cholesterol-modified DNA nanostructures by using a novel surfactant-assisted gel extraction. The addition of sodium cholate (SC) to the sample solution before structure folding prevented aggregation; this was confirmed by gel electrophoresis. We also found that adding sodium dodecyl sulfate (SDS) to the sample inhibited structural folding. The cholesterol-modified DNA nanostructures prepared with SC were successfully purified by gel extraction, and their ability to bind to the lipid membrane surfaces was maintained. This method will facilitate the purification of DNA nanostructures modified with hydrophobic molecules and expand their applicability in the construction of artificial cell-like systems.     

Biomimetic Thermo-Sensitive Hydrogel Encapsulating Hemangiomas Stem Cell Derived Extracellular Vesicles Promotes Microcirculation Reconstruction in Diabetic Wounds

Tissue repair and regeneration in ischemic areas require effective strategies for angiogenesis and microcirculation reconstruction. In this research, extracellular vesicles (EVs) derived from hemangioma stem cells (HemSC) are used as bio-functional materials for angiogenesis. In addition, a novel thermo-sensitive hydrogel based on chitosan (CS) and modified with hyaluronic oligosaccharides (oHA), is specially developed as an ideal carrier of HemSC-EVs. The oHA/CS^gel provides a sustained-release delivery system for EVs, enhances the angiogenic function of HemSC-EVs, and exerts other bio-functions to comprehensively accelerate tissue repair. The physical properties of EVs@oHA/CS^gel proved to be soft, highly elastic deformable, biocompatible, and can maintain the shape and structure of EVs. In vitro co-culture assay verifies the angiogenic effect of EVs@oHA/CS^gel, and this effect is also evaluated in a diabetic wound model. Compared to untreated group, the EVs@oHA/CS^gel group has only 26.9% wound area on day 14, and 226.8% blood flux mark on day 17. Pharmacological mechanisms of HemSC-EVs are predicted by RNA-seq, and cluster analysis of miRNAs predicted targets presents several up-regulated biological processes involving in angiogenesis and wound healing. In conclusion, it is suggested that EVs@oHA/CS^gel as a satisfying therapeutic system for microcirculation reconstruction in tissue repair.