Stem Cell-Derived Extracellular Vesicles for Cancer Therapy and Tissue Engineering Applications.

Brain injury following cardiac arrest (CA) is a significant cause of mortality and poor prognosis in patients, and effective treatment strategies remain limited. Stem cell-derived extracellular vesicles (EVs), a novel cell-free therapeutic approach, have recently demonstrated significant potential in the field of brain injury repair. EVs, key mediators of stem cell paracrine and autocrine signaling, are enriched with bioactive molecules such as non-coding RNAs and proteins. These EVs have the capacity to traverse the bloodstream, reach injury sites, and modulate various biological processes, including neuronal survival, oxidative stress, inflammatory responses, blood-brain barrier integrity, and neurovascular regeneration.This review aims to provide a comprehensive overview of the research history, structural characteristics, and in vivo distribution and metabolism of stem cell-derived EVs. The review further explores their therapeutic potential and underlying mechanisms in post-CA brain injury, including the inhibition of neuronal apoptosis, alleviation of oxidative stress and inflammation, promotion of blood-brain barrier repair, and enhancement of neurovascular regeneration. Additionally, the review highlights emerging directions and challenges in the clinical application of stem cell-derived EVs, offering theoretical insights and perspectives for future research and translational development. The potential of stem cell-derived EVs as a breakthrough strategy for treating post-CA brain injury is underscored, offering renewed optimism for enhancing patient outcomes.

Stem cell-derived extracellular vesicles (EVs) play a crucial role in intercellular communication and reflect the functional characteristics of their parent cells. Despite the significant therapeutic potential of these EVs, the molecular features of EVs derived from different stem cells and their relationship with tissue specificity remain underexplored. In this study, we conducted integrated proteomic and transcriptomic analyses of EVs derived from six dental stem cells (DSCs) and three systemic stem cells (SSCs) to investigate the role of EVs in tissue-specific molecular transfer. This study found that although DSCs and SSCs share 92.1% of the core proteome, the EVs derived from each stem cell type displayed distinct molecular signatures. EVs from DSCs were enriched in signaling molecules, reflecting their parent cells' roles in local tissue repair, whereas EVs from SSCs carried mitochondrial and metabolic proteins, indicating their preferential involvement in metabolic regulation. Furthermore, we uncovered that the parent cells transferred 83% of the core proteins to their EVs in a tissue-specific pattern, ensuring that dental EVs retain prominent signaling functions. Thus, understanding the relationship between EVs and their parent stem cells with respect to tissue origin would be helpful to harness EVs as targeted therapeutic agents, particularly in oral regenerative medicine.

A very important cause of the frustration with drug therapy for central nervous system (CNS) diseases is the failure of drug delivery. The blood-brain barrier (BBB) prevents most therapeutic molecules from entering the brain while maintaining CNS homeostasis. Scientists are keen to develop new brain drug delivery systems to solve this dilemma. Extracellular vesicles (EVs), as a class of naturally derived nanoscale vesicles, have been extensively studied in drug delivery due to their superior properties. This review will briefly present current brain drug delivery strategies, including invasive and non-invasive techniques that target the brain, and the application of nanocarriers developed for brain drug delivery in recent years, especially EVs. The cellular origin of EVs affects the surface protein, size, yield, luminal composition, and other properties of EVs, which are also crucial in determining whether EVs are useful as drug carriers. Stem cell-derived EVs, which inherit the properties of parental cells and avoid the drawbacks of cell therapy, have always been favored by researchers. Thus, in this review, we will focus on the application of stem cell-derived EVs for drug delivery in the CNS. Various nucleic acids, proteins, and small-molecule drugs are loaded into EVs with or without modification and undergo targeted delivery to the brain to achieve their therapeutic effects. In addition, the challenges facing the clinical application of EVs as drug carriers will also be discussed. The directions of future efforts may be to improve drug loading efficiency and precise targeting.

Stem cell-derived extracellular vesicles (EVs) have shown great promise in the field of regenerative medicine and tissue engineering. Recently, human bone marrow-derived mesenchymal stem cell (BMSC)-derived EVs have been considered for bone tissue engineering applications. In this study, we evaluated the osteogenic capability of placental stem cell (PSC)-derived EVs and compared them to the well-characterized BMSC-derived EVs. EVs were extracted from three designated time points (0, 7, and 21 days) after osteogenic differentiation. The results showed that the PSC-derived EVs had much higher protein and lipid concentrations than EVs derived from BMSCs. The extracted EVs were characterized by observing their morphology and size distribution before utilizing next-generation sequencing to determine their microRNA (miRNA) profiles. A total of 306 miRNAs within the EVs were identified, of which 64 were significantly expressed in PSC-derived EVs that related to osteogenic differentiation. In vitro osteogenic differentiation study indicated the late-stage (21-day extracted)-derived EVs higher osteogenic enhancing capability when compared with the early stage-derived EVs. We demonstrated that EVs derived from PSCs could be a new source of EVs for bone tissue engineering applications.

Extracellular vesicles (EVs) are crucial for intercellular communication and affect various physiological and pathological processes. Although terrestrial EVs have been extensively studied, marine-derived EVs have yet to be explored. This study investigated the therapeutic potential of sea cucumbers, known for their regenerative and immune abilities. Sea cucumber extracellular matrix (ECM)-anchored EVs (SEVs) were isolated and characterized using physical and electrophoretic analyses. Morphological assessments have shown that SEVs have shape and size distributions similar to mammalian EVs. Internal cargo analysis revealed the encapsulation of diverse proteins and genetic molecules. In anti-inflammatory tests with a lipopolysaccharide (LPS)-induced macrophage model, the results have shown that SEVs can alleviate inflammation factors regarding inducible nitric oxide synthase (iNOS) protein and immune-related mRNA expression. Microarray analysis was conducted to elucidate SEV's pharmacological efficacy and anti-inflammatory mechanisms, showing that SEVs inhibit the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway. An invivo study using a mouse model of atopic dermatitis (AD) induced by 2,4-dinitrochlorobenzene (DNCB) involved subcutaneous SEV administration, followed by severity scoring and histological analyses. Therapeutic efficacy analysis indicated improvements in the AD mouse models, including reduced skin thickness and mast cell numbers. These findings indicate their potential for treating AD. This study highlights the potential clinical applications of marine-derived EVs and offers important implications for future research and therapeutic developments.

[This retracts the article DOI: 10.21037/sci.2017.08.07.].

Neural stem cells and adult hippocampal neurogenesis modulate synaptic plasticity and cognitive function. Neural stem cells secrete extracellular vesicles - microvesicles carrying biomolecular cargos - that modulate the function of other cells and contribute to homeostasis and plasticity in the central nervous system. Alzheimer's disease is marked by a reduction of neural stem cells in the hippocampus dentate gyrus. While increased neural stem cells often correlate with better learning and memory, neurogenesis alone does not always preserve these processes, indicating that other mechanisms involving neural stem cells support memory. It has been shown that intracerebroventricular delivery of neural stem cell-derived small extracellular vesicles in wild-type mice reduces cognitive decline and toxic oligomer binding to synapses. We hypothesize that adequate neural stem cell numbers support neural stem cell-derived small extracellular vesicles protection of synapses against Alzheimer's disease toxic oligomers. Here, we show that elements of immune response in the central nervous system, particularly microglia, may contribute to this protective effect. Specifically, fluorescent-labeled small extracellular vesicles injected into wildtype mice brains were taken up by microglia, with only neural stem cell-derived small extracellular vesicles causing increased microglial activation, indicated by CD68 immunostaining. RNA-sequencing data showed selective activation of immune pathways in microglia by neural stem cell-derived small extracellular vesicles, leading to greater activation and higher Tau uptake 24 hours post-neural stem cell-derived small extracellular vesicle administration. Single-nuclei RNAsequencing of hippocampal microglia gene revealed modulation related to lysosomal activity, supporting neural stem cell-derived small extracellular vesicleinduced neuroprotection via microglia. This study uncovers a novel mechanism through which neural stem cell-derived small extracellular vesicles enhance microglial activity and provide neuroprotection in the hippocampus. Our data demonstrates that neural stem cell-derived small extracellular vesicle uptake by microglia leads to increased microglial activation and improved uptake of Tau oligomers by microglia, suggesting that neural stem cell-derived small extracellular vesicles may prime microglia for a more effective immune response. These results support the hypothesis that neural stem cell-derived small extracellular vesicle-induced modulation of microglial function is crucial for preserving neuronal integrity and mitigating neurodegenerative processes. By elucidating the interactions between neural stem cell-derived small extracellular vesicles and microglia, our study opens new avenues for developing therapeutic strategies aimed at boosting microglial function and addressing neurodegenerative diseases such as Alzheimer's disease.

Extracellular vesicles (EVs) can deliver many types of drugs with their natural source material transport properties, inherent long-term blood circulation capabilities and excellent biocompatibility, and have great potential in the field of drug carrier. Modification of the content and surface of EVs according to the purpose of treatment has become a research focus to improve the drug load and the targeting of EVs. EVs can maximize the stability of the drugs, prevent immune clearance and achieve accurate delivery. Therefore, EVs can be described as \" stealth transport aircrafts \" for drugs. This chapter will respectively introduce the application of natural EVs as cell substitutes in cell therapy and engineered EVs as carriers of nucleic acids, proteins, small molecule drugs and therapeutic viral particles in disease treatment. It will also explain the drug loading and modification strategies of EVs, the source and characteristics of EVs. In addition, the commercialization progress of EVs drugs will be mentioned here, and the problems in their applications will be discussed in conjunction with the application of EVs in the treatment of COVID-19.

Chapter 5 - Stem Cell Extracellular Vesicles: A Novel Cell-Based Therapy for Cardiovascular Diseases

Extracellular vesicles are lipid bilayer-enclosed extracellular membrane vesicles secreted from cells that have gradually shown promise in cancer treatment. As an essential intercellular communication mediator, extracellular vesicles carry bioactive molecules including proteins, lipids, and nucleic acids, which aid in the control and reprogramming of cell signals and ultimately accelerate the spread of cancer. Simultaneously, extracellular vesicles have become a highly desirable option for drug delivery carriers because of the special capacity to cross boundaries and low immunogenicity. Bypassing the endosomal pathway and lysosomal degradation, they can provide medications directly, increasing their effectiveness and decreasing their negative effects. We review the extracellular vesicles’ development, drug loading techniques, and discuss their potential in clinical applications to provide new avenues for precise and targeted treatment strategies. Additionally, engineering modifications of extracellular vesicles can enhance their ability to target specific cells, improving the stability and effectiveness of cargoes, further driving the development of cancer therapy. In this review, we particularly highlight new therapeutic applications of extracellular vesicles, including the latest progress in extracellular vesicle vaccines in tumors. Nonetheless, there are still a lot of obstacles to be overcome before extracellular vesicles may be used to treat cancer. Future research needs to focus on the characteristics of extracellular vesicles from various sources and explore their specific roles in disease treatment to fully leverage the potential of extracellular vesicles in clinical applications such as drug delivery.

Extracellular vesicles (EVs) are a kind of heterogeneous vesicles released by essentially all cell types, which plays an important role in intercellular communication and in the regulation of physiological process (or pathology). EVs mainly includes microvesicles (50–1000 nm), apoptotic bodies (50–5000 nm) and exosomes (30–150 nm), based on distinct biogenesis pathways. Microvesicles are formed by direct budding of the plasma membrane, in which ARF6 and RhoA are important regulators. In addition, oncosomes are also a kind of microvesicles, which are specifically released by tumor cells and contain many specific molecules of tumor cells. When cells undergo apoptosis, they could also release a population of EVs called apoptotic bodies. This kind of EVs is formed through outward blebbing and fragmentation of the cell membrane. Exosomes are derived from multivesicular bodies (MVBs), and released by cells upon fusion of MVBs with the plasma membrane. Exosomes have complex formation and regulatory mechanisms, including endosomal sorting complex dependent and non-dependent mechanisms. Many researchers have shown that EVs with different types and different cell sources may have different components and functions. EVs mainly carry the bioactive molecules of donor cells, such as proteins, DNA or RNA, and target nearby or remote cells to perform different biological functions, thus affecting the physiological and pathological processes of recipient cells. Cells can also discharge their waste by EVs. By transporting cargoes to extracellular, EVs can regulate the amount of intracellular cargoes, thus affecting the survival status of cells. This approach can be beneficial to cells (removing harmful substances) or harmful (transporting drugs outside the cell). Secondly, some key molecules in EVs can be regulated by donor cells according to their state. So, EVs can also reflect the state changes of donor cells, and have the potential as a biomarker for the diagnosis and treatment of diseases. Due to their stable existence in body fluids and derived from various tissues of the body, EVs can not only be a new biomarker of liquid biopsy, but also reflect the heterogeneity and the genesis or development of diseases. As a natural endogenous material, EVs can also serve as a potential drug delivery carrier, and have the advantages of low immunogenicity and crossing the blood brain barrier. In addition, EVs have many other applications. For example, they can be an alternative to cells as a therapeutic agent, as well as targets of disease treatment. This review summarizes the following parts: Firstly, biological origin, biological composition, biological characteristics, and biological functions of EVs. Secondly, the isolation and characterization of EVs. Thirdly, the application of EVs in the diagnosis and treatment of diseases were introduced. At present, the heterogeneity, biological composition and the formation mechanism of EVs need to be further studied, so that it can be better applied in the diagnosis and treatment of diseases. The effective isolation method is also necessary for its clinical application. It is believed that with the development of various biological technologies, these problems will be gradually solved, the biological function of EVs will be also clearly understood, and EVs based application in diseases will make new breakthroughs.

The therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) in kidney injury has been largely reported. However, new approaches are necessary to optimize the efficacy in the treatment of renal diseases. MSCs physiologically are under a low O2 partial pressure (pO2), and culturing adipose-derived MSCs (ADMSCs) in hypoxia alters their secretory paracrine properties. The aim of this study was to evaluate whether hypoxia preconditioning of ADMSCs alters the properties of secreted EVs to improve renal recovery after ischemia-reperfusion injury (IRI). The supernatants of ADMSCs cultivated under 21% pO2 (control) or 1% pO2 (hypoxia) were ultracentrifuged for EVs isolation that were posteriorly characterized by flow cytometry and electron microscopy. The uptake and effects of these EVs were analyzed by using in vitro and in vivo models. HK-2 renal tubule cell line was submitted do ATP depletion injury model. Proteomic analyses of these cells treated with EVs after injury were performed by nano-UPLC tandem nano-ESI-HDMSE method. For in vivo analyses, male Wistar rats were submitted to 45 min bilateral ischemia, followed by renal intracapsular administration of ADMSC-EVs within a 72 h reperfusion period. Histological, immunohistochemical and qRT-PCR analysis of these kidneys were performed to evaluate cell death, inflammation and oxidative stress. Kidney function was evaluated by measuring the blood levels of creatinine and urea. The results demonstrate that hypoxia increases the ADMSCs capacity to secrete EVs that trigger different energy supply, antiapoptotic, immunomodulatory, angiogenic and anti-oxidative stress responses in renal tissue compared with EVs secreted in normoxia. Proteomic analyses of renal tubule cells treated with EVs from ADMSCs in normoxia and hypoxia give a specific signature of modulated proteins for each type of EVs, indicating regulation of distinct biological processes. In summary, hypoxia potentially offers an interesting strategy to enhance the properties of EVs in the treatment of acute kidney disease.

Development of extracellular vesicles in diagnostics and therapeutics: From extracellular vesicles to precision medicine

Skeletal muscle regeneration relies on the tightly temporallyregulatedlineage progression of muscle stem/progenitor cells (MPCs) from activationto proliferation and, finally, differentiation. However, with aging,MPC lineage progression is disrupted and delayed, ultimately causingimpaired muscle regeneration. Extracellular vesicles (EVs) have attractedbroad attention as next-generation therapeutics for promoting tissueregeneration. As a next step toward clinical translation, strategiesto manipulate EV effects on downstream cellular targets are needed.Here, we developed an engineering strategy to tune the therapeuticpotential of EVs using nanotopographical cues. We found that EVs releasedby young MPCs cultured on flat substrates (fEVs) promoted the proliferationof aged MPCs while EVs released by MPCs cultured on nanogratings (nEVs)promoted myogenic differentiation. We then employed a bioengineered3D muscle aging model to optimize the administration protocol andtest the therapeutic potential of fEVs and nEVs in a high-throughputmanner. We found that the sequential administration first of fEVsduring the phase of MPC proliferative expansion (i.e., 1 day afterinjury) followed by nEV administration at the stage of MPC differentiation(i.e., 3 days after injury) enhanced aged muscle regeneration to asignificantly greater extent than fEVs and nEVs delivered either inisolation or mixed. The beneficial effects of the sequential EV treatmentstrategy were further validated in vivo, as evidencedby increased myofiber size and improved functional recovery. Collectively,our study demonstrates the ability of topographical cues to tune EVtherapeutic potential and highlights the importance of optimizingthe EV administration strategy to accelerate aged skeletal muscleregeneration.

Simple SummaryExtracellular vesicles represent a heterogeneous family of lipid bilayer-enclosed particles naturally released by all cytotypes into the extracellular space. They can act as mediators of cell–cell communication. Recently, the applications of extracellular vesicles (EVs) have been of great interest in translational cancer research. The advances in next-generation omics technologies allow us to discover EVs’ selective cargo packaging and as EV roles in horizontal transport, non-invasive biomarkers, and as new therapeutic options. Herein, discussing recent studies, information on extracellular vesicle (EV) biogenesis, morphological characteristics, isolation, and current detection methods is summarized.In recent years, knowledge of cell-released extracellular vesicle (EV) functions has undergone rapid growth. EVs are membrane vesicles loaded with proteins, nucleic acids, lipids, and bioactive molecules. Once released into the extracellular space, EVs are delivered to target cells that may go through modifications in physiological or pathological conditions. EVs are nano shuttles with a crucial role in promoting short- and long-distance cell–cell communication. Comprehension of the mechanism that regulates this process is a benefit for both medicine and basic science. Currently, EVs attract immense interest in precision and nanomedicine for their potential use in diagnosis, prognosis, and therapies. This review reports the latest advances in EV studies, focusing on the nature and features of EVs and on conventional and emerging methodologies used for their separation, characterization, and visualization. By searching an extended portion of the relevant literature, this work aims to give a summary of advances in nanomedical applications of EVs. Moreover, concerns that require further studies before translation to clinical applications are discussed.