Search Results (42)

Articular cartilage (AC) has a very limited capacity for self-healing once damaged. Chondrocytes maintain AC homeostasis and are key cells in AC tissue engineering (ACTE). However, chondrocytes lose their function due to oxidative stress. Umbilical cord mesenchymal stem cells (UCMSCs) are investigated as an alternative cell source for ACTE. MSCs are known to regulate tissue regeneration through host cell modulation, largely via extracellular vesicle (EV)-mediated cell-to-cell communication. The purpose of this study was to verify whether UCMSC-derived EVs (UCMSC-EVs) enhance chondrocyte function. The mean particle sizes of the UCMSC-EVs were 79.8 ± 19.05 nm. Transmission electron microscopy (TEM) revealed that UCMSC-EVs exhibited a spherical morphology. The presence of CD9, CD63, and CD81 confirmed the identity of UCMSC-EVs, with α-tubulin undetected. UCMSC-EVs maintained chondrocyte survival, and increased chondrocyte proliferation after intake by chondrocytes. UCMSC-EVs upregulated mRNA levels of SOX-9, collagen type II (Col-II), and Aggrecan, while decreasing collagen type I (Col-I) levels. UCMSC-EVs reduced the oxidative stress of chondrocytes by reducing mitochondrial superoxide production and increasing protein levels of SOD-2 and Sirt-3 in chondrocytes. The 50 most abundant known microRNAs (miRNAs) derived from UCMSC-EVs were selected for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. GO analysis revealed enrichment in pathways associated with small GTPase-mediated signal transduction, GTPase regulatory activity, and mitochondrial matrix. The KEGG analysis indicated that these miRNAs may regulate chondrocyte function through the PI3K-Akt, MAPK, and cAMP signaling pathways. In summary, this study shows that UCMSC-EVs enhance chondrocyte function and may be applied to ACTE. Full article

Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) have shown great promise in promoting tissue repair, including skin wound healing, but challenges like rapid degradation and short retention have limited their clinical application. Hydrogels have emerged as effective carriers for sustained EV release. Three-dimensional printing enables the development of personalized skin substitutes tailored to the wound size and shape. This study aimed to develop 3D bioprinted gelatin–genipin hydrogels incorporating human umbilical cord MSC-sEVs (hUCMSC-sEVs) for future skin wound healing applications. Gelatin hydrogels (8% and 10% w/v) were crosslinked with 0.3% genipin (GECL) to improve stability. The hydrogels were evaluated for their suitability for extrusion-based 3D bioprinting and physicochemical properties, such as the swelling ratio, hydrophilicity, enzymatic degradation, and water vapor transmission rate (WVTR). Chemical characterization was performed using EDX, XRD, and FTIR. The hUCMSC-sEVs were isolated via centrifugation and tangential flow filtration (TFF) and characterized. The crosslinked hydrogels were successfully 3D bioprinted and demonstrated superior properties, including high hydrophilicity, a swelling ratio of ~500%, slower degradation, and optimal WVTR. hUCMSC-sEVs, ranging from 50 to 200 nm, were positive for surface and cytosolic markers. Adding 75 μg/mL of hUCMSC-EVs into 10% GECL hydrogels significantly improved the biocompatibility. These hydrogels offer ideal properties for 3D bioprinting and wound healing, demonstrating their potential as biomaterial scaffolds for skin tissue regeneration applications. Full article

Osteoarthritis (OA) is a prevalent joint disorder that lacks effective therapies to halt cartilage degeneration. Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) are being investigated as promising chondroprotective agents. Compared to primary MSCs, induced pluripotent stem cell (iPSC)-derived MSCs (MLCs) offer superior scalability and enhanced paracrine activity. The aim of this study was to explore the feasibility of using autologous MLC-derived sEVs as a potential therapeutic strategy for OA through the analysis of their protein cargo. iPSCs from an OA patient and a healthy donor were differentiated into MLCs. sEVs were isolated from these MLCs and characterized, with a particular focus on their protein cargo. Both iPSC lines were successfully differentiated into MLCs, which secreted sEVs with comparable size distributions and yields. The analysis of differentially expressed proteins revealed a high abundance of proteins associated with OA pathology and cartilage degradation in sEVs from OA MLCs compared to those from healthy MLCs. The persistence of OA-associated protein signatures in autologous MLC-derived sEVs may limit their therapeutic efficacy. These findings underscore the importance of carefully evaluating disease-specific protein profiles in sEVs for regenerative applications. Full article

Sepsis is a risk factor associated with increasing neonatal morbidity and mortality, acute lung injury, and chronic lung disease. While stem cell therapy has shown promise in alleviating acute lung injury, its effects are primarily exerted through paracrine mechanisms rather than local engraftment. Accumulating evidence suggests that these paracrine effects are mediated by mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs), which play a critical role in immune system modulation and tissue regeneration. sEVs contain a diverse cargo of mRNA, miRNA, and proteins, contributing to their therapeutic potential. We hypothesize that sEVs derived from three distinct sources, cord blood plasma (CBP), Wharton jelly (WJ), and placental (PL) MSCs, may prevent the cytotoxicity induced by E. coli lipopolysaccharide (LPS) in lung alveolar epithelial cells. Objective: To determine the effects of CBP-, WJ-, and PL-MSCs-derived sEVs on cell viability, apoptosis, and proinflammatory cytokine production in alveolar epithelial cells and monocytes following LPS treatment. sEVs were collected from conditioned media of PL-MSCs, WJ-MSCs, and CBP using 50 nm membrane filters. sEVs were characterized based on nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting techniques. The protein concentration of isolated sEVs was used to standardize treatment doses. A549 cells and monocyte THP-1 cells were cultured and exposed to LPS in the presence or absence of sEVs for 72 h. Cell viability was measured using CellTiter-Glo 2.0 chemiluminescence-based assay. For cytokine analysis, A549 and THP-1 cells were pre-incubated for 24 h with or without PL- and CBP-sEVs, followed by exposure to LPS or control conditions for an additional 24 h. The conditioned media were collected, and interleukin-6 (IL-6) and interleukin-8 (IL-8) levels were quantified using ELISA. LPS treatment significantly reduced the viability of both A549 and THP-1 cells. The presence of CB- or WJ-sEVs significantly increased cell viability compared to controls. Cells treated with PL-sEVs showed increased cell viability but did not reach statistical significance. LPS-treated cells showed a significant increase in apoptosis and elevated levels of pro-inflammatory cytokines IL-6 and IL-8. All three sEVs types (CBP-, WJ-, and PL-sEVs) significantly reduced LPS-induced apoptosis and IL-6 release. Interestingly, while WJ-sEVs decreased IL-8, both CBP- and PL-sEVs led to an increase in IL-8 compared to their respective controls. CBP-, PL-, and WJ-derived sEVs demonstrated protective effects against LPS-induced injury in alveolar epithelial cells and monocytes, as evidenced by increased cell viability and modulation of pro-inflammatory cytokine release. These findings suggest that placenta-derived sEVs have the potential to modulate the immune response, mitigate inflammation, and prevent end-organ damage in neonatal sepsis. Full article

Mesenchymal stem/stromal cell-derived small extracellular vesicles (MSC-sEVs) are promising therapeutic agents. In this study, we investigated how the administration route of MSC-sEVs affects their therapeutic efficacy in a mouse model of bleomycin (BLM)-induced skin scleroderma (SSc). We evaluated the impact of topical (TOP), subcutaneous (SC), and intraperitoneal (IP) administration of MSC-sEVs on dermal fibrosis, collagen density, and thickness. All three routes of administration significantly reduced BLM-induced fibrosis in the skin, as determined by Masson’s Trichrome staining. However, only TOP administration reduced BLM-induced dermal collagen density, with no effect on dermal thickness observed for all administration routes. Moreover, SC, but not TOP or IP administration, increased anti-inflammatory profibrotic CD163⁺ M2 macrophages. These findings indicate that the administration route influences the therapeutic efficacy of MSC-sEVs in alleviating dermal fibrosis, with TOP administration being the most effective, and this efficacy is not mediated by M2 macrophages. Since both TOP and SC administration target the skin, the difference in their efficacy likely stems from variations in MSC-sEV delivery in the skin. Fluorescence-labelled TOP, but not SC MSC-sEVs when applied to skin explant cultures, localized in the stratum corneum. Hence, the superior efficacy of TOP over SC MSC-sEVs could be attributed to this localization. A comparison of the proteomes of stratum corneum and MSC-sEVs revealed the presence of >100 common proteins. Most of these proteins, such as filaggrin, were known to be crucial for maintaining skin barrier function against irritants and toxins, thereby mitigating inflammation-induced fibrosis. Therefore, the superior efficacy of TOP MSC-sEVs over SC and IP MSC-sEVs against SSc is mediated by the delivery of proteins to the stratum corneum to reinforce the skin barrier. Full article

Interrupted blood flow in the brain due to ischemic injuries such as ischemic stroke or traumatic brain injury results in irreversible brain damage, leading to cognitive impairment associated with inflammation, disruption of the blood–brain barrier (BBB), and cell death. Since the BBB only allows entry to a small class of drugs, many drugs used to treat ischemia in other tissues have failed in brain-related disorders. The administration of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) has shown promise in improving the functional recovery of the brain following cerebral ischemia by inducing blood vessel formation. To facilitate such a treatment approach, it is necessary to develop bioprocesses that can produce therapeutically relevant MSC-EVs in a reproducible and scalable manner. This study evaluated the feasibility of using stirred suspension bioreactors (SSBs) to scale-up the serum-free production of pro-angiogenic MSC-EVs under clinically relevant physioxic conditions. It was found that MSCs grown in SSBs generated EVs that stimulated angiogenesis in cerebral microvascular endothelial cells, supporting the use of SSBs to produce MSC-EVs for application in cerebral ischemia. These properties were impaired at higher cell confluency, outlining the importance of considering the time of harvest when developing bioprocesses to manufacture EV populations. Full article

Background: Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. Methods: The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. Results: Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 10¹² particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. Conclusions: The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity. Full article

White matter injury (WMI) is a common neurological issue in premature-born neonates, often causing long-term disabilities. We recently demonstrated a key beneficial role of Wharton’s jelly mesenchymal stromal cell-derived small extracellular vesicles (WJ-MSC-sEVs) microRNAs (miRNAs) in WMI-related processes in vitro. Here, we studied the functions of WJ-MSC-sEV miRNAs in vivo using a preclinical rat model of premature WMI. Premature WMI was induced in rat pups through inflammation and hypoxia-ischemia. Small EVs were purified from the culture supernatant of human WJ-MSCs. The capacity of WJ-MSC-sEV-derived miRNAs to decrease microglia activation and promote oligodendrocyte maturation was evaluated by knocking down (k.d) DROSHA in WJ-MSCs, releasing sEVs containing significantly less mature miRNAs. Wharton’s jelly MSC-sEVs intranasally administrated 24 h upon injury reached the brain within 1 h, remained detectable for at least 24 h, significantly reduced microglial activation, and promoted oligodendrocyte maturation. The DROSHA k.d in WJ-MSCs lowered the therapeutic capabilities of sEVs in experimental premature WMI. Our results strongly indicate the relevance of miRNAs in the therapeutic abilities of WJ-MSC-sEVs in premature WMI in vivo, opening the path to clinical application. Full article

Intranasal administration is a promising strategy to enhance the delivery of the sEVsomes-based drug delivery system to the central nervous system (CNS). This study aimed to explore central distributive characteristics of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) and underlying pathways. Here, we observed that intranasal MSC-sEVs were rapidly distributed to various brain regions, especially in the subcortex distant from the olfactory bulb, and were absorbed by multiple cells residing in these regions. We captured earlier transportation of intranasal MSC-sEVs into the perivascular space and found an increase in cerebrospinal fluid influx after intranasal administration, particularly in subcortical structures of anterior brain regions where intranasal sEVs were distributed more significantly. These results suggest that the perivascular pathway may underlie the rapid and widespread central delivery kinetics of intranasal MSC-sEVs and support the potential of the intranasal route to deliver MSC-sEVs to the brain for CNS therapy. Full article

In this study, we employed organic and inorganic dyes that have fluorescence under visible or near-infrared light region to stain human umbilical cord (Huc) mesenchymal stem cell (MSC)-, HEK293T cell- and HGC cell-derived small extracellular vesicles (sEVs), and then tracked their fluorescence signals in human gastric cancer xenografted murine models. Several biological characteristics were examined and compared when different dye-stained sEVs in the same tumor model or the same dye-stained sEVs between different tumor models were applied, including sEVs circulation in the blood, biodistribution of sEVs in major organs, and time-dependent tumor accumulation of sEVs. The results demonstrated that distinct tumor accumulation features were presented by sEVs if labeled by different fluorescent dyes, while sEVs derived from different cell lines showed homologous blood circulation and tumor accumulation. To conclude, although fluorescence imaging remains a reliable way to trace sEVs, single staining of sEVs membrane should be obviated in future work when examining the biological fate of sEVs. Full article

Neurotoxic side effects of chemotherapy include deficits in attention, memory, and executive functioning. Currently, there are no FDA-approved therapies. In mice, cisplatin causes long-term cognitive deficits, white matter damage, mitochondrial dysfunction, and loss of synaptic integrity. We hypothesized that MSC-derived small extracellular vesicles (sEVs) could restore cisplatin-induced cognitive impairments and brain damage. Animals were injected with cisplatin intraperitoneally and treated with MSC-derived sEVs intranasally 48 and 96 h after the last cisplatin injection. The puzzle box test (PBT) and the novel object place recognition test (NOPRT) were used to determine cognitive deficits. Synaptosomal mitochondrial morphology was analyzed by transmission electron microscopy. Immunohistochemistry using antibodies against synaptophysin and PSD95 was applied to assess synaptic loss. Black-Gold II staining was used to quantify white matter integrity. Our data show that sEVs enter the brain in 30 min and reverse the cisplatin-induced deficits in executive functioning and working and spatial memory. Abnormalities in mitochondrial morphology, loss of white matter, and synaptic integrity in the hippocampus were restored as well. Transcriptomic analysis revealed upregulation of regenerative functions after treatment with sEVs, pointing to a possible role of axonal guidance signaling, netrin signaling, and Wnt/Ca²⁺ signaling in recovery. Our data suggest that intranasal sEV treatment could become a novel therapeutic approach for the treatment of chemobrain. Full article

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA). Full article

Osteoarthritis (OA) is a degenerative disease of the joints characterized by cartilage damage and severe pain. Despite various pharmacological and surgical interventions, current therapies fail to halt OA progression, leading to high morbidity and an economic burden. Thus, there is an urgent need for alternative therapeutic approaches that can effectively address the underlying pathophysiology of OA. Extracellular Vesicles (EVs) derived from mesenchymal stromal cells (MSCs) represent a new paradigm in OA treatment. MSC-EVs are small membranous particles released by MSCs during culture, both in vitro and in vivo. They possess regenerative properties and can attenuate inflammation, thereby promoting cartilage healing. Importantly, MSC-EVs have several advantages over MSCs as cell-based therapies, including lower risks of immune reactions and ethical issues. Researchers have recently explored different strategies, such as modifying EVs to enhance their delivery, targeting efficiency, and security, with promising results. This article reviews how MSC-EVs can help treat OA and how they might work. It also briefly discusses the benefits and challenges of using MSC-EVs and talks about the possibility of allogeneic and autologous MSC-EVs for medical use. Full article

People living with HIV (PLHIV) are at a higher risk of having cerebrocardiovascular diseases (CVD) compared to HIV negative (HIV^neg) individuals. The mechanisms underlying this elevated risk remains elusive. We hypothesize that HIV infection results in modified microRNA (miR) content in plasma extracellular vesicles (EVs), which modulates the functionality of vascular repairing cells, i.e., endothelial colony-forming cells (ECFCs) in humans or lineage negative bone marrow cells (lin⁻ BMCs) in mice, and vascular wall cells. PLHIV (N = 74) have increased atherosclerosis and fewer ECFCs than HIV^neg individuals (N = 23). Plasma from PLHIV was fractionated into EVs (HIV^posEVs) and plasma depleted of EVs (HIV PL^depEVs). HIV^posEVs, but not HIV PL^depEVs or HIV^negEVs (EVs from HIV^neg individuals), increased atherosclerosis in apoE^−/− mice, which was accompanied by elevated senescence and impaired functionality of arterial cells and lin⁻ BMCs. Small RNA-seq identified EV-miRs overrepresented in HIV^posEVs, including let-7b-5p. MSC (mesenchymal stromal cell)-derived tailored EVs (TEVs) loaded with the antagomir for let-7b-5p (miRZip-let-7b) counteracted, while TEVs loaded with let-7b-5p recapitulated the effects of HIV^posEVs in vivo. Lin⁻ BMCs overexpressing Hmga2 (a let-7b-5p target gene) lacking the 3′UTR and as such is resistant to miR-mediated regulation showed protection against HIV^posEVs-induced changes in lin⁻ BMCs in vitro. Our data provide a mechanism to explain, at least in part, the increased CVD risk seen in PLHIV. Full article