Production Of Extracellular Vesicles Research Articles

Exosomes: A Fluid Biopsy Source for Clinical Interventions of Noncommunicable Diseases Treatment: A Review

Introduction: The beginning of exosome biosynthesis is marked by the emergence of the initial endosomes through the inward splitting of the plasma cell membrane. This process is facilitated through the endosomal categorization complex essential for transport, which is also involved in the production of different extracellular vesicles. Exosomes are naturally occurring nanosized vesicles found in all bodily fluids and can be successfully extracted from preserved biological materials, while maintaining their structural integrity. Methods: The articles published recently in high-quality journals (Science Direct, Springer, Institute of Electrical and Electronics Engineers (IEEE), and Taylor & Francis) indexed in various indexing sources such as Scopus, Web of Science, PubMed, Google Scholar, and so on were collected using keywords such as fluid biopsy (FB) exosomes, endosomes, and noncommunicable diseases (NCDs) treatment, new biomarkers, and treatment. Discussion: In recent years, exosomes have emerged as an exciting option for “FB” that has demonstrated significant potential in the areas of noninvasive medical testing, predictions, as well as tracking responses to therapy for noncommunicable diseases. Nevertheless, specific constraints need to be addressed to expand the application of exosome-based FB as a widely accepted and reliable testing method in typical medical facilities. Conclusion: This review provides a comprehensive overview of our present understanding of exosomes to be an FB method for diagnosing, predicting outcomes, and tracking treatment responses in NCDs. It also discusses the main constraints, innovations in technology, as well as future possibilities of using this application in medical treatment.

Glutamine and serum starvation alters the ATP production, oxidative stress, and abundance of mitochondrial RNAs in extracellular vesicles produced by cancer cells

Induction of autophagy represents an effective survival strategy for nutrient-deprived or stressed cancer cells. Autophagy contributes to the modulation of communication within the tumor microenvironment. Here, we conducted a study of the metabolic and signaling implications associated with autophagy induced by glutamine (Gln) and serum starvation and PI3K/mTOR inhibitor and autophagy inducer NVP-BEZ235 (BEZ) in the head and neck squamous cell carcinoma (HNSCC) cell line FaDu. We compared the effect of these different types of autophagy induction on ATP production, lipid peroxidation, mitophagy, RNA cargo of extracellular vesicles (EVs), and EVs-associated cytokine secretome of cancer cells. Both BEZ and starvation resulted in a decline in ATP production. Simultaneously, Gln starvation enhanced oxidative damage of cancer cells by lipid peroxidation. In starved cells, there was a discernible fragmentation of the mitochondrial network coupled with an increase in the presence of tumor susceptibility gene 101 (TSG101) on the mitochondrial membrane, indicative of the sorting of mitochondrial cargo into EVs. Consequently, the abundance of mitochondrial RNAs (mtRNAs) in EVs released by FaDu cells was enhanced. Notably, mtRNAs were also detectable in EVs isolated from the serum of both HNSCC patients and healthy controls. Starvation and BEZ reduced the production of EVs by cancer cells, yet the characteristic molecular profile of these EVs remained unchanged. We also found that alterations in the release of inflammatory cytokines constitute a principal response to autophagy induction. Importantly, the specific mechanism driving autophagy induction significantly influenced the composition of the EVs-associated cytokine secretome.

Chronic hypoxia for the adaptation of extracellular vesicle phenotype

Variations in oxygen level affect the phenotype of cells and extracellular vesicles (EVs). Depending on the metabolic oxygen demand of cells, hypoxic cell culture can produce conditions more like those found in vivo, and with appropriate oxygen levels, mimic hypoxic tumours. However, most previous experiments studying both EVs and the effects of hypoxia on cells use periods of 72 h or less of hypoxia. We hypothesised that this was insufficient time for adaptation to hypoxic conditions both for EVs and cells which may skew the results of such studies. In this study, the effects of acute (72 h) and chronic hypoxia (> 2 weeks) on the phenotype of HepG2 and PC3 cells and their EVs were examined. Cells could be cultured normally under chronic hypoxic conditions and cryopreserved and recovered. The effects of hypoxia on EV phenotype are slow to establish and dependent on cell line. In PC3 cells, the greatest change in phenotype and increase in EV production occurred only with chronic hypoxic culture. In HepG2 cells, the number of EVs produced was insensitive to hypoxic culture and the greatest changes in protein expression were observed after acute hypoxic culture. Nonetheless, biphasic changes in EV phenotype were detected in both cell types in response to either acute or chronic hypoxia. These results indicate that for cells which do not induce consumptive oxygen depletion, prolonged hypoxic culture is required for complete adaptation.

Ank-mediated pyrophosphate regulates shear stress-induced small extracellular vesicle production in 3D-cultured osteocytes

ABSTRACT Osteocytes are located in the lacunae of fluid-filled bone and communicate with neighboring or distant cells by secreting small extracellular vesicles (sEVs) and growth factors as well as via dendrite–dendrite direct connections. However, the mechanism regulating sEV production in osteocytes is yet to be elucidated. In this study, we investigated sEV production and its underlying mechanism in osteocytes cultured on a three dimensional (3D) scaffold. We employed a perfusion system to apply shear stress stimulation to MLO-Y4 cells cultured on a 3D biphasic calcium phosphate (BCP) scaffold and analyzed sEV production and gene expression using RNA sequencing. We found that the expression of genes associated with sEV biogenesis and the secretory pathway were enhanced by fluid shear stress in MLO-Y4 cells cultured on a 3D BCP scaffold. In particular, fluid shear stress induced the expression of Ank, a pyrophosphate transporter, in 3D-cultured MLO-Y4 cells. The role of Ank in sEV production was further examined. Probenecid, an Ank inhibitor, significantly suppressed shear stress-induced sEV production, whereas Ank cDNA overexpression stimulated it. The inhibition of shear stress-induced sEV production by probenecid was recovered by the exogenous addition of pyrophosphate to MLO-Y4 cells. These findings suggest that shear stress-mediated sEV production in 3D-cultured osteocytes is regulated by extracellular pyrophosphate transported by Ank.

Characterization of MSC Growth, Differentiation, and EV Production in CNF Hydrogels Under Static and Dynamic Cultures in Hypoxic and Normoxic Conditions.

Mesenchymal stem cells (MSCs) hold immense therapeutic potential due to their regenerative and immunomodulatory properties. However, to utilize this potential, it is crucial to optimize their in vitro cultivation conditions. Three-dimensional (3D) culture methods using cell-laden hydrogels aim to mimic the physiological microenvironment in vitro, thus preserving MSC biological functionalities. Cellulosic hydrogels are particularly promising due to their biocompatibility, sustainability, and tunability in terms of chemical, morphological, and mechanical properties. This study investigated the impact of (1) two physical crosslinking scenarios for hydrogels derived from anionic cellulose nanofibers (to-CNF) used to encapsulate adipose-derived MSCs (adMSCs) and (2) physiological culture conditions on the in vitro proliferation, differentiation, and extracellular vesicle (EV) production of these adMSCs. The results revealed that additional Ca2+-mediated crosslinking, intended to complement the self-assembly and gelation of aqueous to-CNF in the adMSC cultivation medium, adversely affected both the mechanical properties of the hydrogel spheres and the growth of the encapsulated cells. However, cultivation under dynamic and hypoxic conditions significantly improved the proliferation and differentiation of the encapsulated adMSCs. Furthermore, it was demonstrated that the adMSCs in the CNF hydrogel spheres exhibited potential for scalable EV production with potent immunosuppressive capacities in a bioreactor system. These findings underscore the importance of physiological culture conditions and the suitability of cellulosic materials for enhancing the therapeutic potential of MSCs. Overall, this study provides valuable insights for optimizing the in vitro cultivation of MSCs for various applications, including tissue engineering, drug testing, and EV-based therapies.

Extracellular Vesicles Contribute to Oxidized LDL-Induced Stromal Cell Proliferation in Benign Prostatic Hyperplasia.

Clinical and experimental evidence has linked Benign Prostatic Hyperplasia (BPH) with dyslipidemic and hypercholesterolemic conditions, though the underlying cellular mechanisms remain unclear. This study investigates the impact of dyslipidemia, specifically oxidized LDL (OxLDL), on prostatic stromal cell proliferation and the release of extracellular vesicles (EVs). Mice were fed a high-fat diet, and human prostatic stromal cells (HPSCs) were treated with OxLDL. Proliferation assays and EV characterization were performed to assess the role of EVs in BPH progression. Pro-atherogenic conditions significantly increased cell proliferation in both murine prostatic cells and HPSCs. Treatment with metformin effectively inhibited OxLDL-induced proliferation. Additionally, OxLDL stimulated the production and release of pro-proliferative EVs by HPSCs, which further promoted cellular proliferation. The findings suggest that dyslipidemia drives prostatic stromal cell proliferation and EV secretion, contributing to BPH progression. Metformin demonstrates potential as a therapeutic agent to mitigate these effects, offering insight into novel strategies for BPH management. This study highlights the complex interaction between dyslipidemia, cell proliferation, and extracellular communication in the context of BPH pathogenesis.

Efficient Treatment of Psoriasis Using Conditioned Media from Mesenchymal Stem Cell Spheroids Cultured to Produce Transforming Growth Factor-β1-Enriched Small-Sized Extracellular Vesicles.

Psoriasis is a common chronic inflammatory disease in which keratinocytes proliferate abnormally due to excessive immune action. Psoriasis can be associated with various comorbidities and has a significant impact on health-related quality of life. Although many systemic treatments, including biologic agents, have been developed, topical treatment remains the main option for psoriasis management. Consequently, there is an urgent need to develop topical treatments with minimal side effects and high efficacy. Mesenchymal stem cells (MSCs) exhibit excellent immune regulation, anti-inflammatory activities, and therapeutic effects, and MSC-derived extracellular vesicles (EVs) can serve as crucial mediators of functional transfer from MSCs. Therefore, this study aimed to develop a safe and easy-to-use emulsion cream for treating psoriasis using MSC conditioned media (CM) containing EVs. We developed an enhanced Wharton's jelly MSC (WJ-MSC) culture method through a three-dimensional (3D) culture containing exogenous transforming growth factor-β3. Using the 3D culture system, we obtained CM from WJ-MSCs, which yielded a higher EV production compared to that of conventional WJ-MSC culture methods, and investigated the effect of EV-enriched 3D-WJ-MSC-CM cream on psoriasis-related inflammation. Administration of the EV-enriched 3D-WJ-MSC-CM cream significantly reduced erythema, thickness, and scaling of skin lesions, alleviated imiquimod-induced psoriasiform lesions in mice, and ameliorated histopathological changes in mouse skin. The upregulated mRNA expression of inflammatory cytokines, including IL-17a, IL-22, IL-23, and IL-36, decreased in the lesions. In conclusion, we present here a new topical treatment for psoriasis using an MSC EV-enriched cream.

Genetically engineered human induced pluripotent stem cells for the production of brain-targeting extracellular vesicles

BackgroundExtracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising, natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture, human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However, hiPSC-EVs only have a moderate ability for brain delivery. Herein, we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor.MethodsAn RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging.ResultsHere we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype, express pluripotency markers, and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways, compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency.ConclusionTaken together, we have established stable, genetically engineered hiPSCs for producing EVs with RVG expression, offering the improved ability for brain-targeted drug delivery.

The Immobilization of an FGF2-Derived Peptide on Culture Plates Improves the Production and Therapeutic Potential of Extracellular Vesicles from Wharton's Jelly Mesenchymal Stem Cells.

The skin is an essential organ that protects the body from external aggressions; therefore, damage from various wounds can significantly impair its function, and effective methods for regenerating and restoring its barrier function are crucial. This study aimed to mass-produce wound-healing exosomes using a fragment of the fibroblast growth factor 2 (FGF2)-derived peptide (FP2) to enhance cell proliferation and exosome production. Our experiments demonstrated increased cell proliferation when Wharton's jelly mesenchymal stem cells (WJ MSCs) were coated with FP2. Exosomes from FP2-coated WJ MSCs were analyzed using nanoparticle-tracking analysis, transmission electron microscopy, and Western blotting. Subsequently, fibroblasts were treated with these exosomes, and their viability and migration effects were compared. Anti-inflammatory effects were also evaluated by inducing pro-inflammatory factors in RAW264.7 cells. The treatment of fibroblasts with FP2-coated WJ MSC-derived exosomes (FP2-exo) increased the expression of FGF2, confirming their wound-healing effect in vivo. Overall, the results of this study highlight the significant impact of FP2 on the proliferation of WJ MSCs and the anti-inflammatory and wound-healing effects of exosomes, suggesting potential applications beyond wound healing.

Delivery of therapeutic RNA by extracellular vesicles derived from Saccharomyces cerevisiae for Medicine Applications

Employing small extracellular vesicles (EVs) as drug delivery vehicles presents a plethora of advantages over conventional drug delivery methods, including biological compatibility, engineering versatility for targeted delivery, and biodegradability. Therefore, strategies aimed at amplifying their therapeutic potential involve developing efficient, tissue-specific, and non-immunogenic delivery approaches. Despite rapid advancements in the realm of EVs as drug delivery systems in recent years, the availability of a high-yield, reproducible, and cost-effective source for EVs production and isolation remains a limiting factor for practical application. In this study, we isolated EVs from Saccharomyces cerevisiae (S.c) and loaded them with cargoes such as hsa-miR-143 (an apoptosis-inducing miRNA) or miR-H6 (a miRNA targeting HSV-1). We demonstrated the capability of these EVs to deliver microRNAs or even large mRNA to a variety of cell types. The therapeutic potential of S.c-derived EVs (S.c-EVs) was further evidenced by their ability to inhibit tumor growth in animal models. The S.c-EVs proved to be safe and non-immunogenic in vivo. Our results suggest that Saccharomyces cerevisiae represents a cost-effective source of extracellular vesicles, serving as nanocarriers for functional drug delivery in therapeutic applications.

Snorkel-tag based affinity chromatography for recombinant extracellular vesicle purification.

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine is still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present the Snorkel-tag, for which we have genetically fused the EV surface marker protein CD81, to a series of tags with an additional transmembrane domain to be displayed on the EV surface, resembling a snorkel. This system enables the affinity purification of EVs from complex matrices in a non-destructive form while maintaining EV characteristics in terms of surface protein profiles, associated miRNA patterns and uptake into a model cell line. Therefore, we consider the Snorkel-tag to be a widely applicable tool in EV research, allowing for efficient preparation of EV standards and reference materials, or dissecting EVs with different surface markers when fusing to other tetraspanins in vitro or in vivo.

Keratinocyte derived extracellular vesicles mediated crosstalk between epidermis and dermis in UVB-induced skin inflammation.

Ultraviolet-B (UVB) light induces dermal inflammation, although it is mostly absorbed in the epidermis. Recent reports suggest extracellular vesicles (EVs) act as a mediator of photodamage signaling. Melatonin is reported to be a protective factor against UV-induced damage. We hypothesized that EVs derived from UVB-irradiated keratinocytes might trigger proinflammatory responses in dermal cells and tested whether melatonin can ameliorate UVB-induced inflammation. We used UVB-irradiated HaCaT cells, primary keratinocytes and STING knock-out mice to model production of EVs under photodamaging conditions and performed immunoblotting and ELISA to measure their effect on dermal macrophages. UVB-irradiated keratinocytes produce an increased number of EVs that contain higher concentrations of DNA and protein compared with controls. KC-derived EVs (KEVs) induced a STING- and inflammasome-mediated proinflammatory response in macrophages in vitro, and a pronounced inflammatory infiltrate in mouse dermis in vivo. Melatonin ameliorated KEVs inflammatory effect both in vitro and in vivo. This data suggests EVs are mediators in a crosstalk that takes place between keratinocytes and their neighboring cells as a result of photodamage. Further studies exploring EVs induced by damaging doses of UVB, and their impact on other cells will provide insight into photodamage and may help develop targeted therapeutic approaches.

Oncolytic viruses alter the biogenesis of tumor extracellular vesicles and influence their immunogenicity

Extracellular vesicles (EVs) are mediators of intercellular communication in the tumor microenvironment. Tumor EVs are commonly associated with metastasis, immunosuppression or drug resistance. Viral infections usually increase EV secretion, but little is known about the effect of oncolytic viruses (OVs) on tumor EVs. Here, we investigated the impact of oncolytic vesicular stomatitis virus (VSV) and vaccinia virus (VACV) on EVs secreted by human melanoma and thoracic cancer cells. We found that OV infection increases the production of EVs by tumor cells. These EVs contain proteins of viral origin, such as VSV-G, thus creating a continuum of particles sharing markers of both canonical EVs and viruses. As such, the presence of VSV-G on EVs improves the transfer of their protein content to cell types commonly found in the tumor microenvironment. A proteomic analysis also revealed that EVs-OV secreted during VSV infection are enriched in immunity-related proteins. Finally, CD8+ T cells incubated with EVs-OV from infected cells display slightly enhanced cytotoxic functions. Taken together, these data suggest that OVs enhance the communication mediated by tumor EVs, which could participate to the therapeutic efficacy of OVs. These results also provide rationale for engineering OVs to exploit EVs and disseminate therapeutic proteins within the tumor microenvironment.

Treatment of neurologic pathology and inflammation in Machado-Joseph disease through in vivo self-assembled siRNA.

Machado-Joseph disease, also known as Spinocerebellar ataxia type 3 (MJD/SCA3), is a fatal autosomal dominant hereditary ataxia characterized by cerebellar ataxia resulting from the abnormal expansion of CAG repeats in exon 10 of the ATXN3 gene. Presently, there is no effective treatment for SCA3. Small interfering RNAs (siRNAs) are emerging as potential therapeutic strategies to specifically target the disease-causing mutant ATXN3 (mATXN3) protein. However, the delivery efficiency of siRNAs remains a major obstacle for clinical application, particularly in brain disorders. This study aimed to develop a synthetic biology strategy to reprogram the host liver as a tissue chassis to induce and deliver in vivo self-assembled siRNAs (IVSA-siRNAs) to target the ATXN3 gene. A synthetic construct directed by a cytomegalovirus promoter was designed to encode a neuron-targeting rabies virus glycoprotein tag and mATXN3-siRNA. After intravenous injection, the synthetic construct was taken up by mouse livers, which were then reprogrammed to enable the self-assembly, production, and secretion of small extracellular vesicles (sEVs) encapsulating mATXN3-siRNA. The sEV-encapsulated mATXN3-siRNA was further transported through the endogenous circulating system of sEVs, crossing the blood-brain barrier and reaching the cerebellar cortex and spinal cerebellar tract, where they silenced the ATXN3 gene. Treatment with the synthetic construct for 8 or 12 weeks led to significant improvements in motor balance ability and reduction of cerebellar atrophy in YACMJD84.2 transgenic mice. The number of Purkinje cells in the cerebellar cortex was significantly increased, and the loss of myelin basic protein was reduced. Moreover, the quantity of neurotoxic nuclear inclusion bodies and the expression of glial fibrillary acidic protein, which promotes neuroinflammation in activated astrocytes, were decreased significantly. The synthetic construct facilitated the generation and delivery of IVSA-siRNA to the cerebellar cortex and spinal cerebellar tract, thereby inhibiting the expression of mATXN3 protein. This treatment successfully addressed motor impairments, alleviated neuropathological phenotypes, and mitigated neuroinflammation in YACMJD84.2 transgenic mice. Our strategy effectively overcomes the primary challenges associated with siRNA therapy for cerebellar ataxia, offering a promising avenue for future clinical treatments.

Enhanced Extracellular Vesicle Cargo Loading via microRNA Biogenesis Pathway Modulation.

Extracellular vesicles (EVs) are physiological vectors for the intercellular transport of a variety of molecules. Among these, small RNAs, and especially microRNAs (miRNAs), have been identified as prevalent components, and there has thus been a robust investigation of EVs for therapeutic miRNAs delivery. However, intrinsic levels of EV-associated miRNAs are generally too low to enable efficient and effective therapeutic outcomes. We hypothesized that miRNA localization to EVs could be improved by limiting competing interactions that occur throughout the miRNA biogenesis process. Using miR-146a-5p as a model, modulation of transcription, nuclear export, and enzymatic cleavage steps of miRNA biogenesis were tested for impact on EV miRNA loading. Working in HEK293T cells, various alterations in the EV biogenesis pathway were shown to impact miRNA localization to EVs. The system was then applied in induced pluripotent stem cells (iPSCs), a more promising substrate for therapeutic EV production, and EVs were separated and assessed for anti-inflammatory efficacy in vitro and in a murine colitis model, where the preservation of function was validated. Overall, the results highlight necessary considerations when designing a cell culture system for the devoted production of miRNA-loaded EVs.

Exploration of microRNAs from blood extracellular vesicles as biomarkers of exposure to polycyclic aromatic hydrocarbons

Exposure to polycyclic aromatic hydrocarbons (PAHs), ubiquitously environmental contaminant, leads to the development of major toxic effects on human health, such as carcinogenic and immunosuppressive alterations reported for the most studied PAH, i.e., benzo(a)pyrene (B(a)P). In order to assess the risk associated with this exposure, it is necessary to have predictive biomarkers. Thus, extracellular vesicles (EVs) and their microRNA (miRNA) contents, have recently been proposed as potentially interesting biomarkers in Toxicology. Our study here explores the use of vesicles secreted and found in blood fluids, and their miRNAs, as biomarkers of exposure to B(a)P alone and within a realistic occupational mixture. We isolated EVs from primary human cultured blood mononuclear cells (PBMCs) and rat plasma after PAH exposure and reported an increased EV production by B(a)P, used either alone or in the mixture, in vitro and in vivo. We then investigated the association of this EV release with the blood concentration of the 7,8,9,10-hydroxy (tetrol)-B(a)P reactive metabolite, in rats. By performing RNA-sequencing (RNA-seq) of miRNAs in PBMC-derived EVs, we analyzed miRNA profiles and demonstrated the regulation of the expression of miR-342–3p upon B(a)P exposure. We then validated B(a)P-induced changes of miR-342–3p expression in vivo in rat plasma-derived EVs. Overall, our study highlights the feasibility of using EVs and their miRNA contents, as biomarkers of PAH exposure and discusses their potential in environmental Toxicology.

Improvement of androgenic alopecia by extracellular vesicles secreted from hyaluronic acid-stimulated induced mesenchymal stem cells

BackgroundAndrogenetic alopecia (AGA) is a common form of hair loss. Androgens, such as testosterone and dihydrotestosterone, are the main causes of AGA. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can reduce AGA. However, preparing therapeutic doses of MSCs for clinical use is challenging. Induced pluripotent stem cell-derived MSCs (iMSCs) are homogenous and easily expandable, enabling scalable production of EVs. Hyaluronic acid (HA) can exert various functions including free radical scavenging, immune regulation, and cell migration. Herein, we examined whether hyaluronic acid (HA) stimulation of iMSCs could produce EVs with enhanced therapeutic outcomes for AGA.MethodsEVs were collected from iMSCs primed with HA (HA–iMSC–EVs) or without HA (iMSC–EVs). The characteristics of EVs were examined using dynamic light scattering, cryo-transmission electron microscopy, immunoblotting, flow cytometry, and proteomic analysis. In vitro, we compared the potential of EVs in stimulating the survival of hair follicle dermal papilla cells undergoing testosterone-mediated AGA. Additionally, the expression of androgen receptor (AR) and relevant growth factors as well as key proteins of Wnt/β-catenin signaling pathway (β-catenin and phosphorylated GSK3β) was analyzed. Subsequently, AGA was induced in male C57/BL6 mice by testosterone administration, followed by repeated injections of iMSC–EVs, HA–iMSC–EVs, finasteride, or vehicle. Several parameters including hair growth, anagen phase ratio, reactivation of Wnt/β-catenin pathway, and AR expression was examined using qPCR, immunoblotting, and immunofluorescence analysis.ResultsBoth types of EVs showed typical characteristics for EVs, such as size distribution, markers, and surface protein expression. In hair follicle dermal papilla cells, the mRNA levels of AR, TGF-β, and IL-6 increased by testosterone was blocked by HA–iMSC–EVs, which also contributed to the augmented expression of trophic genes related to hair regrowth. However, no notable changes were observed in the iMSC–EVs. Re-activation of Wnt/β-catenin was observed in HA–iMSC–EVs but not in iMSC–EVs, as shown by β-catenin stabilization and an increase in phosphorylated GSK3β. Restoration of hair growth was more significant in HA–iMSC–EVs than in iMSC–EVs, and was comparable to that in mice treated with finasteride. Consistently, the decreased anagen ratio induced by testosterone was reversed by HA–iMSC–EVs, but not by iMSC–EVs. An increased expression of hair follicular β-catenin protein, as well as the reduction of AR was observed in the skin tissue of AGA mice receiving HA–iMSC–EVs, but not in those treated with iMSC–EVs.ConclusionsOur results suggest that HA–iMSC–EVs have potential to improve AGA by regulating growth factors/cytokines and stimulating AR-related Wnt/β-catenin signaling.

Urine-derived stem cells serve as a robust platform for generating native or engineered extracellular vesicles

BackgroundMesenchymal stromal cell (MSC) therapy holds great potential yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome that includes extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect both yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to enhance potency, increase circulation time or mediate targeting. However, efficient transfection of MSCs is challenging. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production.MethodsWe isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to genetically engineer USCs using transposon technology.ResultsThere were no differences between time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the assessed time points, however, male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 h without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell cultures were established.ConclusionsIsolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

Mechanical force of uterine occupation enables large vesicle extrusion from proteostressed maternal neurons.

Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate the extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here, we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.

Mechanical force of uterine occupation enables large vesicle extrusion from proteostressed maternal neurons

Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate the extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here, we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.