Therapeutic potential of extracellular vesicles.

Extracellular vesicles are released by all cell types and contain proteins, microRNAs, mRNAs, and other bioactive molecules. Extracellular vesicles play an important role in intercellular communication and in the modulation of the immune system and neuroinflammation. The cargo of extracellular vesicles (e.g., proteins and microRNAs) is altered in pathological situations. Extracellular vesicles contribute to the pathogenesis of many pathologies associated with sustained inflammation and neuroinflammation, including cancer, diabetes, hyperammonemia and hepatic encephalopathy, and other neurological and neurodegenerative diseases. Extracellular vesicles may cross the blood-brain barrier and transfer pathological signals from the periphery to the brain. This contributes to inducing neuroinflammation and cognitive and motor impairment in hyperammonemia and hepatic encephalopathy and in neurodegenerative diseases. The mechanisms involved are beginning to be understood. For example, increased tumor necrosis factor α in extracellular vesicles from plasma of hyperammonemic rats induces neuroinflammation and motor impairment when injected into normal rats. Identifying the mechanisms by which extracellular vesicles contribute to the pathogenesis of these diseases will help to develop new treatments and diagnostic tools for their easy and early detection. In contrast, extracellular vesicles from mesenchymal stem cells have therapeutic utility in many of the above pathologies, by reducing inflammation and neuroinflammation and improving cognitive and motor function. These extracellular vesicles recapitulate the beneficial effects of mesenchymal stem cells and have advantages as therapeutic tools: they are less immunogenic, may not differentiate to malignant cells, cross the blood-brain barrier, and may reach more easily target organs. Extracellular vesicles from mesenchymal stem cells have beneficial effects in models of ischemic brain injury, Alzheimer’s and Parkinson’s diseases, hyperammonemia, and hepatic encephalopathy. Extracellular vesicles from mesenchymal stem cells modulate the immune system, promoting the shift from a pro-inflammatory to an anti-inflammatory state. For example, extracellular vesicles from mesenchymal stem cells modulate the Th17/Treg balance, promoting the anti-inflammatory Treg. Extracellular vesicles from mesenchymal stem cells may also act directly in the brain to modulate microglia activation, promoting a shift from a pro-inflammatory to an anti-inflammatory state. This reduces neuroinflammation and improves cognitive and motor function. Two main components of extracellular vesicles from mesenchymal stem cells which contribute to these beneficial effects are transforming growth factor-β and miR-124. Identifying the mechanisms by which extracellular vesicles from mesenchymal stem cells induce the beneficial effects and the main molecules (e.g., proteins and mRNAs) involved may help to improve their therapeutic utility. The aims of this review are to summarize the knowledge of the pathological effects of extracellular vesicles in different pathologies, the therapeutic potential of extracellular vesicles from mesenchymal stem cells to recover cognitive and motor function and the molecular mechanisms for these beneficial effects on neurological function.

Acute and chronic neurodegenerative conditions (NDs) are major causes of disability and mortality worldwide. Acute NDs encompass conditions such as stroke, traumatic brain injury (TBI), and spinal cord injury (SCI). On the other hand, chronic NDs include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). Currently, no definitive cure exists for these diseases, and available therapies focus primarily on slowing the progression of symptoms. Mesenchymal stem cells (MSCs), due to their multilineage differentiation capacity, immunomodulatory abilities, and regenerative properties, have gained attention in regenerative medicine. In recent years, extracellular vesicles (EVs) derived from MSCs have shown great promise as a cell-free therapeutic approach, eliminating the risks associated with direct MSCs use, such as tumorigenicity and poor cell survival after transplantation. EVs have emerged as powerful mediators of intercellular communication and tissue repair, exhibiting immunomodulatory, anti-inflammatory, and proregenerative properties. However, limitations such as low EVs yield and reduced efficacy due to MSCs replicative senescence restrict their therapeutic potential. Preconditioning strategies, including hypoxia, 3D cultures, and biochemical priming, have been explored in other fields to enhance EVs properties, yet their specific application to NDs remains under-reported. This review aims to address this gap by analyzing the preconditioning methods used to boost the therapeutic potential of MSCs-derived EVs for neurodegenerative diseases. These preconditioning strategies may enhance EVs yield, functional cargo, and targeted therapeutic efficacy for treating acute and chronic NDs.

Mesenchymal cells were described for their potency to interact with human immune cells and to modulate thereby immune responses. Besides mesenchymal stromal cells (MSC) from diverse tissue sources, like bone marrow and umbilical cord, also mesenchymal adherent cells within the heart tissue were able to attenuate and modulate induced immune cell activation or inflammatory processes. Mechanistic studies with MSC support the hypothesis, that mainly paracrine acting molecules, in particular extracellular vesicles (EVs), are responsible for the observed functional effects. EVs are known as potent intercellular communicators by delivering proteins, lipids, RNA and other small signaling molecules to a recipient cell. They can be discriminated by their size and biogenesis into the subsets of apoptotic bodies (diameter > 1μm), microvesicles (diameter range = 1 - 0.1 μm) and exosomes (diameter < 0.1 μm). While microvesicles and apoptotic bodies are shedded from the plasma membrane, exosomes originate from intracellular located multivesicular bodies, which have to fuse with the plasma membrane for their release into the extracellular space. Crosstalk of EVs from MSCs with immune cells is a secured fact, but the way of up-take into the target cells and especially the mechanism of immune modulation are not entirely understood. In our study, we isolated and characterized EVs from a human mesenchymal cardiac cell type regarding their potential to modulate induced immune responses in vitro. The presence of typical EV surface markers like tetraspanins (CD9, CD63, CD81) was confirmed as well as their low expression of HLA-molecules, which indicates a general low immunogenicity. Furthermore, EVs were able to attenuate triggered T cell proliferation accompanied by significantly reduced levels of pro-inflammatory cytokines (IFNγ, TNFα), which was highly dependent on the presence of CD14-positive cells. Interestingly, the EVs of cardiac mesenchymal cells induced a changed phenotype on these myeloid cells; most prominently a reduced HLA-DR and CD86 expression, but enhanced levels for CD206 and PD-L1. Future studies have to identify key molecular pathways involved in EVs` crosstalk with immune cells and to estimate the benefits but also the risks of this new therapeutic based on mesenchymal cells.

Besides being powerhouses of the cell, mitochondria released into extracellular space act as intercellular signaling. Mitochondria and their components mediate cell-to-cell communication in free form or embedded in a carrier. The pathogenesis of cardiovascular disease is complex, which shows close relationship with inflammation and metabolic abnormalities. Since mitochondria sustain optimal function of the heart, extracellular mitochondria are emerging as a key regulator in the development of cardiovascular disease. In this review, we provide recent findings in the presence and forms of mitochondria transfer between cells, as well as the effects of these mitochondria on vascular inflammation and ischemic myocardium. Mitochondrial transplantation is a novel treatment paradigm for patients suffering from acute cardiovascular accident and challenges the traditional methods of mitochondria isolation.

Extracellular Vesicles - A Versatile Biomaterial.

What's in a Name?

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Purpose To assess the therapeutic potential of extracellular vesicles (EVs) derived from stem cells and ocular tissues as a cell-free alternative to traditional stem cell therapies for a broad spectrum of ocular diseases. Methods A comprehensive literature review was performed, focusing on preclinical studies involving EVs derived from mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), neural progenitor cells, immune cells, and ocular-resident cells. Data were extracted on EV cellular origin, isolation methods, routes of administration, preclinical disease models, therapeutic outcomes, and proposed mechanisms of action. Registered clinical trials were also evaluated. Results EVs exhibited regenerative and immunomodulatory effects across a range of ocular conditions, including dry eye, uveitis, glaucoma, retinal degenerations, and optic neuropathies. Various cell sources have been explored for EV production, including MSCs, iPSCs, hESCs, retinal organoids, and other ocular tissue-resident cells. In addition, bioengineered EVs have been developed to modify surface properties or enhance therapeutic cargo. Reported mechanisms of action include miRNA-mediated gene regulation, immune modulation, and oxidative stress reduction. Several early-phase clinical trials are currently underway to translate these findings into human therapies. Conclusion Stem cell-derived EVs represent a promising next-generation, cell-free regenerative therapy for ocular diseases. While preclinical data are promising, successful clinical translation will require optimal EV source selection, scalable and GMP-compliant production, identification of disease-relevant mechanisms of action, rigorous cargo characterization, and alignment with regulatory standards.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

Neural diseases such as compressive, congenital, and traumatic injuries have diverse consequences, from benign mild sequelae to severe life-threatening conditions with associated losses of motor, sensory, and autonomic functions. Several approaches have been adopted to control neuroinflammatory cascades. Traditionally, mesenchymal stem cells (MSCs) have been regarded as therapeutic agents, as they possess growth factors and cytokines with potential anti-inflammatory and regenerative effects. However, several animal model studies have reported conflicting outcomes, and therefore, the role of MSCs as a regenerative source for the treatment of neural pathologies remains debatable. In addition, issues such as heterogeneity and ethical issues limited their use as therapeutic agents. To overcome the obstacles associated with the use of traditional agents, we explored the therapeutic potentials of extracellular vesicles (EVs), which contain nucleic acids, functional proteins, and bioactive lipids, and play crucial roles in immune response regulation, inflammation reduction, and cell-to-cell communication. EVs may surpass MSCs in size issue, immunogenicity, and response to the host environment. However, a comprehensive review is required on the therapeutic potential of EVs for the treatment of neural pathologies. In this review, we discuss the action mechanism of EVs, their potential for treating neural pathologies, and future perspectives regarding their clinical applications.

Osteoarthritis (OA), a joint disease, burdens global healthcare due to aging and obesity. Recent studies show that extracellular vesicles (EVs) from bone marrow-derived mesenchymal stem cells (BMSCs) contribute to joint homeostasis and OA management. However, the impact of donor age on BMSC-derived EV efficacy remains underexplored. In this study, we investigated EV efficacy from young BMSCs (2-month-old) in mitigating OA, contrasting them with EVs from aged BMSCs (27-month-old). The study used destabilisation of the medial meniscus (DMM) surgery on mouse knee joints to induce accelerated OA. Cartilage degeneration markers and senescence markers' expression levels were investigated in response to EV treatment. The therapeutic impact of EVs on chondrocytes under inflammatory responses was also evaluated. Despite having similar morphologies, EVs from young BMSCs markedly decreased senescence and improved chondroprotection by activating the PTEN pathway while simultaneously suppressing the upregulation of the PI3K/AKT pathways, proving to be more effective than those from older BMSCs invitro. Furthermore, intraperitoneal injections of EVs from young donors significantly mitigated OA progression by preserving cartilage and reducing synovitis in a surgical OA model using DMM in mice. These findings highlight that donor age as a critical determinant in the therapeutic potential of BMSC-derived EVs for clinical use in OA treatment.

Stem-cell-derived extracellular vesicles (EVs) have emerged as a promising therapeutic option, addressing the limitations of conventional stem cell therapies. However, the variability and poorly defined therapeutic contents of EVs produced under standard 2-dimensional culture conditions present challenges for their clinical application. In this study, we investigated how the therapeutic properties of mesenchymal stem cell (MSC)-derived EVs can be enhanced by culturing MSCs within 3-dimensional hydrogels that have tunable mechanical properties. Our results demonstrate that different mechanical cues from the culture environment can induce specific gene expression changes in MSCs without compromising their inherent characteristics. Furthermore, EVs derived from these MSCs exhibited distinct angiogenic and immunomodulatory activities, which were dependent on the mechanical properties of the hydrogels used. A comprehensive analysis of the cytokines and microRNAs present in the EVs provided additional validation of these findings. By utilizing a noninvasive culture method that eliminates the need for genetic modification or exogenous biochemical supplementation, our approach presents a novel platform for the tailored production of EVs, thereby enhancing their therapeutic potential in regenerative medicine.

New therapeutic options for liver cirrhosis are needed. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising tool for delivering therapeutic factors in regenerative medicine. Our aim is to establish a new therapeutic tool that employs EVs derived from MSCs to deliver therapeutic factors for liver fibrosis. EVs were isolated from supernatants of adipose tissue MSCs, induced-pluripotent-stem-cell-derived MSCs, and umbilical cord perivascular cells (HUCPVC-EVs) by ion exchange chromatography (IEC). To produce engineered EVs, HUCPVCs were transduced with adenoviruses that code for insulin-like growth factor 1 (AdhIGF-I-HUCPVC-EVs) or green fluorescent protein. EVs were characterized by electron microscopy, flow cytometry, ELISA, and proteomic analysis. We evaluated EVs’ antifibrotic effect in thioacetamide-induced liver fibrosis in mice and on hepatic stellate cells in vitro. We found that IEC-isolated HUCPVC-EVs have an analogous phenotype and antifibrotic activity to those isolated by ultracentrifugation. EVs derived from the three MSCs sources showed a similar phenotype and antifibrotic potential. EVs derived from AdhIGF-I-HUCPVC carried IGF-1 and showed a higher therapeutic effect in vitro and in vivo. Remarkably, proteomic analysis revealed that HUCPVC-EVs carry key proteins involved in their antifibrotic process. This scalable MSC-derived EV manufacturing strategy is a promising therapeutic tool for liver fibrosis.

Synovial Mesenchymal Stem Cell-Derived EV-Packaged miR-31 Downregulates Histone Demethylase KDM2A to Prevent Knee Osteoarthritis

Cells convey information among one another. One instrument employed to transmit data and constituents to specific (target) cells is extracellular vesicles (EVs). They originate from a variety of cells (endothelial, immune cells, platelets, mesenchymal stromal cells, etc.), and consequently, their surface characteristics and cargo vary according to the paternal cell. The cargo could be DNA, mRNA, microRNA, receptors, metabolites, cytoplasmic proteins, or pathological molecules, as a function of which EVs exert different effects upon endocytosis in recipient cells. Recently, EVs have become important participants in a variety of pathologies, including atherogenesis and coronavirus disease 2019 (COVID-19)-associated thrombosis. Herein, we summarize recent advances and some of our own results on the role of EVs in atherosclerotic cardiovascular diseases, and discuss their potential to function as signaling mediators, biomarkers and therapeutic agents. Since COVID-19 patients have a high rate of thrombotic events, a special section of the review is dedicated to the mechanism of thrombosis and the possible therapeutic potential of EVs in COVID-19-related thrombosis. Yet, EV mechanisms and their role in the transfer of information between cells in normal and pathological conditions remain to be explored.