Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However, the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs), with unique design features, facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress, using aggregate- and microcarrier-based culture systems.

Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling, flow cytometry, and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blotting. Lipidomics provided insights into EV lipid composition, while functional assays assessed the impact of EVs in a D-galactose-induced senescence model.

VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size, but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36%) in total lipid content; however, distinct differences were identified in lipid chain lengths and saturation levels, affecting key pathways such as sphingolipid and neurotrophin signaling. Human BVO EVs demonstrated the abilities of reducing oxidative stress and increasing cell proliferation in vitro.

Human BVOs differentiated in VWBRs (in particular 40 rpm) produce 2-3 fold higher yield of EVs (per mL) than static control. The bio manufactured EVs from VWBRs have exosomal characteristics and therapeutic cargo, showing functional properties in in vitro assays. This innovative approach establishes VWBRs as a scalable platform for producing functional EVs with defined lipid profiles and therapeutic potential, paving the way for future in vivo studies.

Cellular senescence is intricately linked with numerous changes observed in the aging process, including the depletion of the stem cell pool and the decline in tissue and organ functions. Over the past three decades, efforts to halt and reverse aging have intensified, bringing rejuvenation closer to reality. Current strategies involve treatments using stem cells or their derivatives, such as the secretome. This article aims to highlight key points and evaluate the utilization of secretome derived from mesenchymal stromal cells (MSCs) as an antisenescent approach. Employing a quasi-systematic research approach, the authors conducted a comprehensive analysis based on a search algorithm targeting the in vitro effects of MSC-derived secretome on rescuing cells from a senescent state. Reviewing 39 articles out of 687 hits retrieved from PubMed and Scopus without a time limit, the authors synthesized information and identified common types of MSC-tissue sources utilized (including bone marrow-MSCs, umbilical cord-MSCs, iPSC-derived MSCs, adipose tissue-MSCs, dental pulp-MSCs, amniotic membrane-MSCs, placenta-MSCs, gingival-MSCs, urine-MSCs, and commercially available MSC lineages) from both human and other species (such as mice and rats). The authors also examined the forms of secretome tested (including conditioned media and extracellular vesicles), the cell types treated (MSCs or other cell types), methods/biomarkers of monitoring senescence/rejuvenation, and the mechanisms involved. Ultimately, this review underscores the proof-of-principle of the beneficial effects of MSC-derived secretome in reversing cellular senescence across various cell types. Such insights might aid the scientific community in designing improved in vitro and in vivo assays for future research and clinical validation of this promising cell-free therapy.

Stem cell-derived extracellular vesicles (EVs) offer a promising cell-free approach for cardiovascular regenerative medicine. In this study, we developed magnetically labeled induced pluripotent stem cell-derived EVs (magneto-iPSC-EVs) encapsulated with superparamagnetic iron oxide (SPIO) nanoparticles for image-guided regenerative treatment of myocardial infarction, in which EVs that can be detected by both magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). iPSC-EVs were isolated, characterized per MISEV2023 guidelines, and loaded with SuperSPIO20 nanoparticles using optimized electroporation conditions (300 V, 2 × 10 ms pulses), achieving a high loading efficiency of 1.77 ng Fe/106 EVs. In vitro results show that magneto-iPSC-EVs can be sensitively detected by MPI and MRI, with a detectability of approximately 107 EVs. In a mouse myocardial ischemia-reperfusion model, intramyocardially injected magneto-iPSC-EVs (2 × 109) were imaged non-invasively by in vivo MPI for 7 days and ex vivo MRI, with the presence of magneto-iPSC-EVs confirmed by Prussian blue staining. Therapeutically, both native and magneto- iPSC-EVs significantly improved cardiac function, with a 37.3% increase in left ventricular ejection fraction and 61.0% reduction in scar size. This study highlights the potential of magneto-iPSC-EVs as a cell-free approach for cardiovascular regenerative medicine, offering both non-invasive imaging capabilities and therapeutic benefits for myocardial repair.

To investigate the role of small extracellular vesicles derived from induced pluripotent stem cells (iPSC-sEVs) in periodontal tissue regeneration, elucidate their potential molecular mechanisms, and provide theoretical guidance for the clinical application of iPSC-sEVs as a cell-free therapeutic strategy for periodontal tissue regeneration.

We investigated the effects of iPSC-sEVs on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. The regenerative potential of iPSC-sEVs was evaluated in vivo, using a periodontal defect model. Bulk RNA sequencing was performed to elucidate the underlying molecular mechanisms.

iPSC-sEVs were isolated, characterized, and systemically evaluated for regenerative potential. The results revealed that treatment with iPSC-sEVs significantly enhanced the proliferation, migration, and osteogenic differentiation of PDLSCs. In situ treatment with iPSC-sEVs loaded onto collagen sponges was performed in a rat model of periodontal defects. Micro-CT and histological analyses indicated that iPSC-sEV treatment markedly promoted alveolar bone repair and periodontal ligament regeneration. Mechanistically, the analysis of bulk RNA sequencing data coupled with experimental validation revealed that iPSC-sEV treatment significantly activated the mitogen-activated protein kinase (MAPK) signaling pathway in PDLSCs. Further investigation showed that the inhibition of this pathway completely abolished the proliferative effects of iPSC-sEVs on PDLSCs.

iPSC-sEVs promote PDLSC proliferation through MAPK signaling pathway activation, while also enhancing PDLSC migratory and osteogenic differentiation capacities, facilitates the repair and regeneration of damaged periodontal tissue and presents a potential novel therapeutic strategy for clinical periodontal tissue regeneration.

This review highlights the key molecular and cellular mechanisms contributing to aging, such as DNA damage, mitochondrial dysfunction, telomere shortening, protein dysfunction, and defective autophagy. These biological mechanisms are involved in various oral health conditions prevalent in the elderly, including periodontal disease, oral cancer, xerostomia, dental caries, and temporomandibular joint disorders. Exosomes generated by mesenchymal stem cells possess substantial therapeutic potential. These exosomes are nanosized extracellular vesicles derived from cells and are involved in essential intercellular communication and tissue homeostasis. The exosome-based therapies proved superior to traditional cell-based approaches, due to lower immunogenicity, ease of storage, and avoidance of complications associated with cell transplantation. Furthermore, the diagnostic potential of exosomes as non-invasive biomarkers for aging processes and age-related oral diseases offers insights into disease diagnosis, staging, and monitoring. Among the challenges and future perspectives of translating exosome research from preclinical studies to clinical applications is the need for standardized procedures to fully harness the therapeutic and diagnostic capabilities of exosomes.

This study investigates the potential of extracellular vesicles (EVs) in skin regeneration and rejuvenation. EVs, nanoscale vesicles released by various cell types, play a crucial role in intercellular communication.

To reaffirm the pivotal role of blood-derived exosomes in intercellular communication and their potential for skin tissue regeneration, leveraging existing research, including human data, to advocate for exosomes as a viable cell-free therapy for skin health.

The study employs a novel isolation technique combining PEG and Dextran with ultracentrifugation to extract EVs from plasma. Characterization techniques, including NTA, AFM, Cryo-TEM, and FC, confirm the successful isolation and characterization of EVs.

The study demonstrates positive effects of blood-derived EVs on fibroblast proliferation, collagen, and elastin production in murine and human models. Despite advancements, challenges persist in obtaining consistent EVs quality and concentration. The findings support the clinical relevance of EVs in skin health and suggest potential applications for skin rejuvenation. Future research directions and study limitations are also discussed, contributing to the evolving understanding of EVs-based therapies.

The senescence of bone marrow mesenchymal stem cells (BMMSCs) is increasingly recognized as a critical factor contributing to the pathophysiology of age-related diseases. Recent studies suggest that small extracellular vesicles (sEVs) derived from the serum of elderly individuals may play a pivotal role in promoting BMMSC senescence. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane glycoprotein, is upregulated during cellular senescence and can regulate stem cell ageing. However, the precise mechanisms by which GPNMB influences BMMSCs senescence remain poorly understood. Understanding this relationship could provide valuable insights into therapeutic strategies for enhancing BMMSCs function and mitigating age-related degeneration.

In this study, we conducted comprehensive in vitro experiments to elucidate the effects of sEVs isolated from the serum of elderly donors on the senescence of BMMSCs. We employed advanced proteomic analysis to quantify the expression levels of GPNMB in both BMMSCs and sEVs. Statistical methods were utilized to investigate the correlations between GPNMB expression, glycosylation modifications, and established senescence markers.

Our findings demonstrate a robust positive correlation between the expression of GPNMB in BMMSCs and sEVs and the induction of cellular senescence. Notably, we observed that elevated levels of GPNMB, particularly those bearing bisecting N-acetylglucosamine (GlcNAc) modifications, significantly enhance the senescent phenotype of BMMSCs. Furthermore, we identified the bisecting GlcNAc modification at the Asn 249 residue of GPNMB as a critical determinant for its senescence-promoting function.

This study elucidates the substantial role of sEVs derived from mesenchymal stem cells in exacerbating BMMSC senescence through mechanisms that are critically dependent on the presence of bisecting GlcNAcylated GPNMB. These insights emphasize the necessity of targeting glycosylation modifications of GPNMB in the design of novel senolytic therapies aimed at mitigating cellular ageing and its associated pathologies.

Age is the most important risk factor for degenerative diseases such as osteoarthritis (OA). It is associated with the accumulation of senescent cells in joint tissues that contribute to the pathogenesis of OA, in particular through the release of senescence-associated secretory phenotype (SASP) factors. Mesenchymal stromal cells (MSCs) and their derived extracellular vesicles (EVs) are promising treatments for OA. However, the senoprotective effects of MSC-derived EVs in OA have been poorly investigated. Here, we used EVs from human adipose tissue-derived MSCs (ASC-EVs) in two models of inflammaging (IL1β)- and DNA damage (etoposide)-induced senescence in OA chondrocytes. We showed that the addition of ASC-EVs was effective in reducing senescence parameters, including the number of SA-β-Gal-positive cells, the accumulation of γH2AX foci in nuclei and the secretion of SASP factors. In addition, ASC-EVs demonstrated therapeutic efficacy when injected into a murine model of OA. Several markers of senescence, inflammation and oxidative stress were decreased shortly after injection likely explaining the therapeutic efficacy. In conclusion, ASC-EVs exert a senoprotective function both in vitro, in two models of induced senescence in OA chondrocytes and, in vivo, in the murine model of collagenase-induced OA.

Rotator cuff tears (RCT) are the most common cause of shoulder pain among adults. "Rotator cuff" refers to the four muscles that cover the shoulder joint: supraspinatus, infraspinatus, subscapularis, and teres minor. These muscles help maintain the rotational movement and stability of the shoulder joint. RCT is a condition in which one or more of these four muscles become ruptured or damaged, causing pain in the arms and shoulders. RCT results from degenerative changes caused by chronic inflammation of the tendons and consequent tendon tissue defects. This phenomenon occurs because of the exhaustion of endogenous tendon stem cells. Tendon regeneration requires rejuvenation of these endogenous tendon stem/progenitor cells (TSPCs) prior to their growth phase. TSPCs exhibit clonogenicity, multipotency, and self-renewal properties; they express classical stem cell markers and genes associated with the tendon lineage. However, specific markers for TSPC are yet to be identified. In this review, we introduce novel TSPC markers and discuss various strategies for TSPC reprogramming. With further research, TSPC reprogramming technology could be adapted to treat age-related degenerative diseases, providing a new strategy for regenerative medicine.

Osteoarthritis (OA) is a common joint degenerative disease which currently lacks satisfactory disease-modifying treatments. Oxidative stress-mediated senescent chondrocytes accumulation is closely associated with OA progression, which abrogates cartilage metabolism homeostasis by secreting senescence-associated secretory phenotype (SASP) factors. Numerous studies suggested mesenchymal stem cells-derived small extracellular vesicles (MSC-sEVs) have been regarded as promising candidates for OA therapy. However, MSC-sEVs were applied before the occurrence of cartilage degeneration or at early-stage OA, while in clinical practice, most OA patients who present with pain are already in non-early-stage. Recently, embryonic stem cells-derived sEVs (ESC-sEVs) have been reported to possess powerful anti-aging effects. However, whether ESC-sEVs could attenuate non-early-stage OA progression remains unknown. In this study, we demonstrated ESC-sEVs ameliorated senescent phenotype and cartilage destruction in both mechanical stress-induced non-early-stage posttraumatic OA and naturally aged mice. More importantly, we found ESC-sEVs alleviated senescent phenotype by rejuvenating aged chondrocytes but not inducing apoptosis. We also provided evidence that the FOXO1A-autophagy axis played an important role in the anti-aging effects of ESC-sEVs. To promote clinical translation, we confirmed ESC-sEVs reversed senescent phenotype in ex-vivo cultured human end-stage OA cartilage explants. Collectively, our findings reveal that ESC-sEVs-based therapy is of high translational value in non-early-stage OA treatment.

Angiogenesis plays a crucial role in the growth of colorectal cancer (CRC). Recent studies have identified extracellular vesicles (EVs) in the tumor microenvironment as important mediators of cell-to-cell communication. However, the specific role and mechanisms of CRC-derived EVs in regulating tumor angiogenesis remain to be further investigated.

EVs were isolated from the conditioned medium of the CRC cells using ultracentrifugation. We investigated the effects of HT-29-derived EVs on tumor growth and angiogenesis in a subcutaneous HT-29 CRC tumor model in mice. Additionally, we evaluated the impact of HT-29-derived EVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, bioinformatics analysis was performed to identify relevant signaling pathways, and pathway inhibitors were used to block the activation of these pathways, aiming to elucidate their roles in angiogenesis.

We found that HT-29-derived EVs can promote tumor growth and angiogenesis in vivo, as well as significantly enhance the proliferation, migration, and tube formation of HUVECs. Bioinformatics analysis revealed that HT-29-derived EVs may regulate angiogenesis through the JAK/STAT3 signaling pathway. Specifically, we observed that CRC-derived EVs promoted the phosphorylation of STAT3 (p-STAT3) and the expression of VEGFA in the nucleus of HUVECs. Treatment with the STAT3 inhibitor Stattic reduced the nuclear expression of p-STAT3, which impaired its function as a transcription factor, thereby inhibiting VEGFA expression and the pro-angiogenic effects of CRC-derived EVs.

EVs derived from CRC cells promote CRC tumor angiogenesis by regulating VEGFA through the JAK/STAT3 pathway in endothelial cells.

Extracellular 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.

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. 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.

Here 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.

Taken together, we have established stable, genetically engineered hiPSCs for producing EVs with RVG expression, offering the improved ability for brain-targeted drug delivery.

Extracellular vesicles (EVs) are membrane-enclosed particles that have attracted much attention for their potential in disease diagnosis and therapy. However, the clinical translation is limited by the dosing consistency due to their heterogeneity. Among various characterization techniques, nanoparticle tracking analysis (NTA) offers distinct benefits for EV characterization. In this review, we will discuss the NTA technique with a focus on factors affecting the results; then, we will review the two modes of the NTA techniques along with suitable applications in specific areas of EV studies. EVs are typically characterized by their size, size distribution, concentration, protein markers, and RNA cargos. The light-scattering mode of NTA offers accurate size, size distribution, and concentration information in solution, which is useful for comparing EV isolation methods, storage conditions, and EV secretion conditions. In contrast, fluorescent mode of NTA allows differentiating EV subgroups based on specific markers. The success of fluorescence NTA heavily relies on fluorescent tags (e.g., types of dyes and labeling methods). When EVs are labeled with disease-specific markers, fluorescence NTA offers an effective tool for disease detection in biological fluids, such as saliva, blood, and serum. Finally, we will discuss the limitations and future directions of the NTA technique in EV characterization.

Apoptosis may disrupt differentiation of hematopoietic stem cells (HSCs), which can affect aging. Thus, the main goal of this study was to compare the effect of plasma exosomes from young and old people on the expression of Bcl-2-associated X (BAX) and B-cell lymphoma 2 (BCL-2) genes in the HSCs. Plasma samples were acquired from four elderly adults and four younger adults, referring to Blood Transfusion Organization of Tehran-Iran during August 2022- September 2022.Then, the exosomes of the samples were extracted and analyzed using DLS, TEM, and CD63 surface marker. HSCs were isolated from umbilical cord blood cells. The MTT test was used to assess the viability of exosomes-treated HSCs at doses of 5 and 10 μg/ml. The expression of BAX and BCL-2 genes in the cells was examined using real-time PCR. A one-way analysis of variance (ANOVA) was performed to examine the distinctions among five groups. The viability of HSCs was not affected by the exosomes from young and old people than the control group (P = 0.453). Exosomes from young people (doses 5 and 10 µg/ml) did not have any significant impacts on BAX (P = 0.746, and P = 0.345, respectively) and BCL-2 (P = 0.773, and P = 0.461, respectively) expression in the HSCs compared to the control group. The BAX gene was significantly upregulated and the BCL-2 gene was significantly downregulated after utilizing the exosomes derived from the plasma of elderly individuals (dose 10 µg/ml) compared to the control (P = 0.001, P = 0.002, respectively). The current research shows that aged people's exosomes can increase BAX/ BCL-2 ratio in umbilical cord blood-derived HSCs compared to control and young groups.

Developing an intracellular delivery system is of key importance in the expansion of protein-based therapeutics acting on cytosolic or nuclear targets. Recently, extracellular vesicles (EVs) have been exploited as next-generation delivery modalities due to their natural role in intercellular communication and biocompatibility. However, fusion of protein of interest to a scaffold represents a widely used strategy for cargo enrichment in EVs, which could compromise the stability and functionality of cargo. Herein, we report intracellular delivery via EV-based approach (IDEA) that efficiently packages and delivers native proteins both in vitro and in vivo without the use of a scaffold. As a proof-of-concept, we applied the IDEA to deliver cyclic GMP-AMP synthase (cGAS), an innate immune sensor. The results showed that cGAS-carrying EVs activated interferon signaling and elicited enhanced antitumor immunity in multiple syngeneic tumor models. Combining cGAS EVs with immune checkpoint inhibition further synergistically boosted antitumor efficacy in vivo. Mechanistically, scRNA-seq demonstrated that cGAS EVs mediated significant remodeling of intratumoral microenvironment, revealing a pivotal role of infiltrating neutrophils in the antitumor immune milieu. Collectively, IDEA, as a universal and facile strategy, can be applied to expand and advance the development of protein-based therapeutics.

Currently, there is a growing focus on aging and age-related diseases. The processes of aging are based on cell senescence, which results in changes in intercellular communications and pathological alterations in tissues. In the present study, we investigate the influence of senescent mesenchymal stem cells (MSCs) on endothelial cells (ECs). In order to induce senescence in MSCs, we employed a method of stress-induced senescence utilizing mitomycin C (MmC). Subsequent experiments involved the interaction of ECs with MSCs in a coculture or the treatment of ECs with the secretome of senescent MSCs. After 48 h, we assessed the EC state. Our findings revealed that direct interaction led to a decrease in EC proliferation and migratory activity of the coculture. Furthermore, there was an increase in the activity of the lysosomal compartment, as well as an upregulation of the genes P21, IL6, IL8, ITGA1, and ITGB1. Treatment of ECs with the "senescent" secretome resulted in less pronounced effects, although a decrease in proliferation and an increase in ICAM-1 expression were observed. The maintenance of high levels of typical "senescent" cytokines and growth factors after 48 h suggests that the addition of the "senescent" secretome may have a prolonged effect on the cells. It is noteworthy that in samples treated with the "senescent" secretome, the level of PDGF-AA was higher, which may explain some of the pro-regenerative effects of senescent cells. Therefore, the detected changes may underlie both the negative and positive effects of senescence. The findings provide insight into the effects of cell senescence in vitro, where many of the organism's regulatory mechanisms are absent.

Mesenchymal stem cells (MSCs) are extensively researched for therapeutic applications in tissue engineering and show significant potential for clinical use. Intrinsic or extrinsic factors causing senescence may lead to reduced proliferation, aberrant differentiation, weakened immunoregulation, and increased inflammation, ultimately limiting the potential of MSCs. It is crucial to comprehend the molecular pathways and internal processes responsible for the decline in MSC function due to senescence in order to devise innovative approaches for rejuvenating senescent MSCs and enhancing MSC treatment. We investigate the main molecular processes involved in senescence, aiming to provide a thorough understanding of senescence-related issues in MSCs. Additionally, we analyze the most recent advancements in cutting-edge approaches to combat MSC senescence based on current research. We are curious whether the aging process of stem cells results in a permanent "memory" and if cellular reprogramming may potentially revert the aging epigenome to a more youthful state.

Cellular senescence (CS) is recognized as one of the hallmarks of aging, and an important player in a variety of age-related pathologies. Accumulation of senescent cells can promote a pro-inflammatory and pro-cancerogenic microenvironment. Among potential senotherapeutics are extracellular vesicles (EVs) (40-1000 nm), including exosomes (40-150 nm), that play an important role in cell-cell communications. Here, we review the most recent studies on the impact of EVs derived from stem cells (MSCs, ESCs, iPSCs) as well as non-stem cells of various types on CS and discuss potential mechanisms responsible for the senotherapeutic effects of EVs. The analysis revealed that (i) EVs derived from stem cells, pluripotent (ESCs, iPSCs) or multipotent (MSCs of various origin), can mitigate the cellular senescence phenotype both in vitro and in vivo; (ii) this effect is presumably senomorphic; (iii) EVs display cross-species activity, without apparent immunogenic responses. In summary, stem cell-derived EVs appear to be promising senotherapeutics, with a feasible application in humans.

Heart failure (HF) is characterized by oxidative stress and mitochondrial dysfunction. This study investigates the therapeutic potential of Necrostatin-1 (Nec-1) delivered through exosomes derived from induced pluripotent stem cells (iPSCs) to address these pathologies in HF.

An HF rat model was established, and comprehensive assessments were performed using echocardiography, hemodynamics, and ventricular mass index measurements. iPSCs were used to isolate exosomes, loaded with Nec-1, and characterized for efficient delivery into cardiomyocytes. The interaction between Nec-1-loaded exosomes (Nec-1-Exos), poly (ADP-ribose) polymerase 1 (PARP1), and apoptosis-inducing factor mitochondria-associated 1 (AIFM1) was explored. Gain-of-function experiments assessed changes in cardiomyocyte parameters, and histological analyses were conducted on myocardial tissues.

Cardiomyocytes successfully internalized Nec-1-loaded exosomes, leading to downregulation of PARP1, inhibition of AIFM1 nuclear translocation, increased ATP and superoxide dismutase levels, reduced reactive oxygen species and malonaldehyde levels, and restored mitochondrial membrane potential. Histological examinations confirmed the modulation of the PARP1/AIFM1 axis by Nec-1, mitigating HF.

iPSC-derived exosomes carrying Nec-1 attenuate oxidative stress and mitochondrial dysfunction in HF by targeting the PARP1/AIFM1 axis. This study proposes a promising therapeutic strategy for HF management and highlights the potential of exosome-mediated drug delivery.

Extracellular vesicles (EVs) are an emergent next-generation biotechnology with broad application potential. In particular, immunomodulatory bioactivity of EVs leading to anti-inflammatory effects is well-characterized. Cell source and culture conditions are critical determinants of EV therapeutic efficacy, while augmenting EV anti-inflammatory bioactivity via diverse strategies, including RNA cargo loading and protein surface display, has proven effective. Yet, translational challenges remain. Additionally, the potential of direct antimicrobial EV functionality has only recently emerged but offers the possibility of overcoming drug-resistant bacterial and fungal infections through novel, multifactorial mechanisms. As discussed herein, these application areas are brought together by the potential for synergistic benefit from technological developments related to EV cargo loading and biomanufacturing.

Extracellular vesicles (EVs) have been proven to play a significant immunoregulatory role in many chronic diseases, such as cancer and immune disorders. Among them, EVs derived from NK cells are an essential component of the immune cell functions. These EVs have been demonstrated to carry a variety of toxic proteins and nucleic acids derived from NK cells and play a therapeutic role in diseases like malignancies, liver fibrosis, and lung injury. However, natural NK-derived EVs (NKEVs) have certain limitations in disease treatment, such as low yield and poor targeting. Concurrently, NK cells exhibit characteristics of memory-like NK cells, which have stronger proliferative capacity, increased IFN-γ production, and enhanced cytotoxicity, making them more advantageous for disease treatment. Recent research has shifted its focus towards engineered extracellular vesicles and their potential to improve the efficiency, specificity, and safety of disease treatments. In this review, we will discuss the characteristics of NK-derived EVs and the latest advancements in disease therapy. Specifically, we will compare different cellular sources of NKEVs and explore the current status and prospects of memory-like NK cell-derived EVs and engineered NKEVs.

Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.

Studies have reported promising results of mesenchymal stem cell therapies for skin aging. However, in the use of mesenchymal stem cells, some drawbacks including rarely possible tumorigenicity and low engraftment rates have limited their widespread clinical use. Adipose tissue stem cell-derived exosomes (ASCEs) are emerging as effective cell-free therapeutic agents.

It was evaluated the clinical efficacy of combining the application of human ASCE-containing solution (HACS) with microneedling to treat facial skin aging.

A 12-week, prospective, randomized, split-face, comparative study was conducted. Twenty-eight individuals underwent three treatment sessions separated by 3-week intervals and were followed up for 6 weeks after the last session. At each treatment session, HACS and microneedling were administered to one side of the face, and normal saline solution and microneedling were administered to the other side as a control.

The Global Aesthetic Improvement Scale score was significantly higher on the HACS-treated side than on the control side at the final follow-up visit (p = 0.005). Objective measurements obtained by different devices including PRIMOS Premium, Cutometer MPA 580, Corneometer CM 825, and Mark-Vu confirmed greater clinical improvements in skin wrinkles, elasticity, hydration, and pigmentation on the HACS-treated side than on the control side. The results of the histopathological evaluation were consistent with the clinical findings. No serious adverse events were observed.

These findings demonstrate that combined treatment using HACS and microneedling is effective and safe for treating facial skin aging.

The term extracellular vesicles (EVs) refers to a variety of heterogeneous nanovesicles secreted by almost all cell types, primarily for intercellular communication and maintaining cellular homeostasis. The role of EVs has been widely reported in the genesis and progression of multiple pathological conditions, and these vesicles are suggested to serve as 'liquid biopsies'. In addition to their use as biomarkers, EVs secreted by specific cell types, especially with stem cell properties, have shown promise as cell-free nanotherapeutics. Stem cell-derived EVs (SC-EVs) have been increasingly used as an attractive alternative to stem cell therapies and have been reported to promote regeneration of aging-associated tissue loss and function. SC-EVs treatment ameliorates brain and peripheral aging, reproductive dysfunctions and inhibits cellular senescence, thereby reversing several aging-related disorders and dysfunctions. The anti-aging therapeutic potential of SC-EVs depends on multiple factors, including the type of stem cells, the age of the source stem cells, and their physiological state. In this review, we briefly describe studies related to the promising effects of SC-EVs against various aging-related pathologies, and then we focus in-depth on the therapeutic benefits of SC-EVs against Alzheimer's disease, one of the most devastating neurodegenerative diseases in elderly individuals. Numerous studies in transgenic mouse models have reported the usefulness of SC-EVs in targeting the pathological hallmarks of Alzheimer's disease, including amyloid plaques, neurofibrillary tangles, and neuroinflammation, leading to improved neuronal protection, synaptic plasticity, and cognitive measures. Cell culture studies have further identified the underlying molecular mechanisms through which SC-EVs reduce amyloid beta (Aβ) levels or shift microglia phenotype from pro-inflammatory to anti-inflammatory state. Interestingly, multiple routes of administration, including nasal delivery, have confirmed that SC-EVs could cross the blood-brain barrier. Due to this, SC-EVs have also been tested to deliver specific therapeutic cargo molecule/s (e.g., neprilysin) to the brain. Despite these promises, several challenges related to quality control, scalability, and biodistribution remain, hindering the realization of the vast clinical promise of SC-EVs.

Extracellular vesicles (EVs) can be released from most cells in the body and act as intercellular messengers transferring information in their cargo to affect cellular function. A growing body of evidence suggests that a subset of EVs, referred to here as 'small extracellular vesicles' (sEVs), can accelerate or slow the processes of ageing and age-related diseases dependent on their molecular cargo and cellular origin. Continued exploration of the vast complexity of the sEV cargo aims to further characterise these systemic vehicles that may be targeted to ameliorate age-related pathologies. Marked progress in the development of mass spectrometry-based technologies means that it is now possible to characterise a significant proportion of the proteome of sEVs (surface and cargo) via unbiased proteomics. This information is vital for identifying biomarkers and the development of sEV-based therapeutics in the context of ageing. Although exercise and physical activity are prominent features in maintaining health in advancing years, the mechanisms responsible are unclear. A potential mechanism by which plasma sEVs released during exercise could influence ageing and senescence is via the increased delivery of cargo proteins that function as antioxidant enzymes or inhibitors of senescence. These have been observed to increase in sEVs following acute and chronic exercise, as identified via independent interrogation of high coverage, publicly available proteomic datasets. Establishing tropism and exchange of functionally active proteins by these processes represents a promising line of enquiry in implicating sEVs as biologically relevant mediators of the ageing process.

Extracellular vesicles (EVs) are small, membrane-bound vesicles used by cells to deliver biological cargo such as proteins, mRNA, and other biomolecules from one cell to another, thus inducing a specific response in the target cell and are a powerful method of cell to cell and organ to organ communication, especially during the pathogenesis of human disease. Thus, EVs may be utilized as prognostic and diagnostic biomarkers, but they also hold therapeutic potential just as mesenchymal stem cells have been used in therapeutics. However, unmodified EVs exhibit poor targeting efficacy, leading to the necessity of engineered EVS. To highlight the advantages and therapeutic promises of engineered EVs, in this review, we summarized the research progress on engineered EVs in the past ten years, especially in the past five years, and highlighted their potential applications in therapeutic development for human diseases. Compared to the existing stem cell-derived EV-based therapeutic strategies, engineered EVs show greater promise in clinical applications: First, engineered EVs mediate good targeting efficacy by exhibiting a targeting peptide that allows them to specifically target a specific organ or even cell type, thus avoiding accumulation in undesired locations and increasing the potency of the treatment. Second, engineered EVs can be artificially pre-loaded with any necessary biomolecular cargo or even therapeutic drugs to treat a variety of human diseases such as cancers, neurological diseases, and cardiovascular ailments. Further research is necessary to improve logistical challenges in large-scale engineered EV manufacturing, but current developments in engineered EVs prove promising to greatly improve therapeutic treatment for traditionally difficult to treat diseases.

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been explored in clinical trials for treatment of diseases with complex pathophysiologies. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, it is initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, it is found that their vascularization bioactivity is similar and their anti-inflammatory bioactivity is superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial is employed. In this in vivo model, iPSC EVs more effectively mediate inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.

Liver fibrosis was initially considered to be an irreversible process which will eventually lead to the occurrence of liver cancer. So far there has been no effective therapeutic approach to treat liver fibrosis although scientists have put tremendous efforts into the underlying mechanisms of this disease. Therefore, in-depth research on novel and safe treatments of liver fibrosis is of great significance to human health. Pluripotent stem cells (PSCs) play important roles in the study of liver fibrosis due to their unique features in self-renewal ability, pluripotency, and paracrine function. This article mainly reviews the applications of PSCs in the study of liver fibrosis in recent years. We discuss the role of PSC-derived liver organoids in the study of liver fibrosis, and the latest research advances on the differentiation of PSCs into hepatocytes or macrophages. We also highlight the importance of exosomes of PSCs for the treatment of liver fibrosis.

The fibrogenic process plays a significant pathophysiological role in the progression of chronic kidney disease. Inhibition of the renin-angiotensin system (RAS) is one strategy to delay disease progression but does not reverse established fibrosis. In this context, induced pluripotent stem cells (iPSCs) have been considered an alternative due to their regenerative potential. iPSCs exert their effects through paracrine signaling, which releases specific biomolecules into the extracellular environment, either directly or within extracellular vesicle (EVs), that can reach target cells. This study aims to evaluate the potential beneficial effects of iPSC-derived EVs (EV-iPSCs) in an in vitro model of fibrosis using mouse mesangial cells (MMCs) stimulated with TGF-β. EV-iPSCs were obtained by differentially ultracentrifuging iPSCs culture medium. MMCs were stimulated with 5 ng/mL of TGF-β and simultaneously treated with or without EV-iPSCs for 24 h. Markers of inflammation, fibrosis, and RAS components were assessed using RT-PCR, western blotting, and immunofluorescence. Under TGF-β stimulus, MMCs exhibited increased expression of inflammation markers, RAS components, and fibrosis. However, these changes were mitigated in the presence of EV-iPSCs. EV-iPSCs effectively reduced inflammation, RAS activation, and fibrogenesis in this fibrosis model involving mesangial cells, suggesting their potential as a strategy to reduce glomerular sclerosis.

The regeneration of weight-bearing bone defects and critical-sized cartilage defects remains a significant challenge. A wide range of nano-biomaterials are available for the treatment of bone/cartilage defects. However, their poor compatibility and biodegradability pose challenges to the practical applications of these nano-based biomaterials. Natural biomaterials inspired by the cell units (e.g., nucleic acids and proteins), have gained increasing attention in recent decades due to their versatile functionality, compatibility, biodegradability, and great potential for modification, combination, and hybridization. In the field of bone/cartilage regeneration, natural nano-based biomaterials have presented an unparalleled role in providing optimal cues and microenvironments for cell growth and differentiation. In this review, we systematically summarize the versatile building blocks inspired by the cell unit used as natural nano-based biomaterials in bone/cartilage regeneration, including nucleic acids, proteins, carbohydrates, lipids, and membranes. In addition, the opportunities and challenges of natural nano-based biomaterials for the future use of bone/cartilage regeneration are discussed.

Human mesenchymal stem cells (MSCs) are therapeutic for clinical applications because of their excellent immunomodulatory and multiple lineage differentiation abilities at tissue injury sites. However, insufficient number of cells and lack of regenerative properties during in vitro expansion still limit the clinical applicability of MSC therapies. Here, we demonstrated a preconditioning strategy with trophoblast stem cell-derived extracellular vesicles (TSC-EVs) to boost the proliferation and regenerative capacity of MSCs.

We employed cell proliferation analyses such as CCK8 and BrdU assays to determine the proliferation-promoting role of TSC-EVs on MSCs. Osteogenic effects of TSC-EVs on MSCs were assessed by alkaline phosphatase (ALP) activity, calcium assays, and calvarial bone defect animal models. For skin regenerative effects, skin wound mice model was exploited to analyze wound-healing rate in this study, as well as immunofluorescence and histological staining evaluates. We also performed the small RNA profiling and RNA-sequencing analyzes to understand the cellular mechanism of TSC-EVs on MSCs.

TSC-EVs significantly promoted MSC proliferation under xeno-free conditions and facilitated the therapeutic effects of MSCs, including osteogenesis, anti-senescence, and wound healing. Transcriptomic analysis also provided evidence that specific microRNAs in TSC-EVs and differentially expressed genes (DEGs) in TSC-EV-treated MSCs showed the possibility of TSC-EVs triggering the regenerative abilities of MSCs with cytokine interaction. Hence, we found that NGF/Akt signaling mediated the regenerative effects of TSC-EVs on MSCs as a particular cellular signaling pathway.

The results of this study demonstrated the functional properties of TSC-EVs on MSCs for MSC-based therapeutic applications, suggesting that TSC-EVs may serve as a potential preconditioning source for MSC therapy in the clinical field of regenerative medicine.

The skin undergoes the formation of fine lines and wrinkles through the aging process; also, burns, trauma, and other similar circumstances give rise to various forms of skin ulcers. Induced pluripotent stem cells (iPSCs) have become promising candidates for skin healing and rejuvenation due to not stimulating inflammatory responses, low probability of immune rejection, high metabolic activity, good large-scale production capacity and potentials for personalized medicine. iPSCs can secrete microvesicles (MVs) containing RNA and proteins responsible for the normal repairing process of the skin. This study aimed to evaluate the possibility, safety and effectiveness of applying iPSCs-derived MVs for skin tissue engineering and rejuvenation applications. The possibility was assessed using the evaluation of the mRNA content of iPSC-derived MVs and the behavior of fibroblasts after MV treatment. Investigating the effect of microvesicle on stemness potential of mesenchymal stem cells was performed for safety concerns. In vivo evaluation of MVs was done in order to investigate related immune response, re-epithelialization and blood vessel formation to measure effectiveness. Shedding MVs were round in shape distributed in the range from 100 to 1000 nm in diameter and positive for AQP3, COL2A, FGF2, ITGB, and SEPTIN4 mRNAs. After treating dermal fibroblasts with iPSC-derived MVs, the expressions of collagens Iα1 and III transcripts (as the main fibrous extracellular matrix (ECM) proteins) were upregulated. Meanwhile, the survival and proliferation of MV treated fibroblasts did not change significantly. Evaluation of stemness markers in MV treated MSCs showed negligible alteration. In line with in vitro results, histomorphometry and histopathology findings also confirmed the helpful effect of MVs in skin regeneration in the rat burn wound models. Conducting more investigations on hiPSCs-derived MVs may lead to produce more efficient and safer biopharmaceutics for skin regeneration in the pharmaceutical market.

Compromised regeneration resulting from the deactivation of Wnt/β-catenin signaling contributes to the progression of chronic obstructive pulmonary disease (COPD) with limited therapeutic options. Extracellular cytokine-induced Wnt-based signaling provides an alternative option for COPD treatment. However, the hydrophobic nature of Wnt proteins limits their purification and use. This study devises a strategy to deliver the membrane-bound wingless-type MMTV integration site family, member 3A (Wnt3a) over a long distance by anchoring it to the surface of extracellular vesicles (EVs). The newly engineered Wnt3aWG EVs are generated by co-expressing Wnt3a with two genes encoding the membrane protein, WLS, and an engineered glypican, GPC6ΔGPI -C1C2. The bioactivity of Wnt3aWG EVs is validated using a TOPFlash assay and a mesoderm differentiation model of human pluripotent stem cells. Wnt3aWG EVs activate Wnt signaling and promote cell growth following human alveolar epithelial cell injury. In an elastase-induced emphysema model, impaired pulmonary function and enlarged airspace are greatly restored by the intravenous delivery of Wnt3aWG EVs. Single-cell RNA sequencing-based analyses further highlight that Wnt3aWG EV-activated regenerative programs are responsible for its beneficial effects. These findings suggest that EV-based Wnt3a delivery represents a novel therapeutic strategy for lung repair and regeneration after injury.

Ageing is a universal and unavoidable phenomenon that significantly increases the risk of developing chronic kidney disease (CKD). It has been reported that ageing is associated with functional disruption and structural damage to the kidneys. Extracellular vesicles (EVs), which are nanoscale membranous vesicles containing lipids, proteins, and nucleic acids, are secreted by cells into the extracellular spaces. They have diverse functions such as repairing and regenerating different forms of ageing-related CKD and playing a crucial role in intercellular communication. This paper reviews the etiology of ageing in CKD, with particular attention paid to the roles of EVs as carriers of ageing signals and anti-ageing therapeutic strategies in CKD. In this regard, the double-edged role of EVs in ageing-related CKD is examined, along with the potential for their application in clinical settings.

Extracellular vesicles (EVs) are cell-derived biological nanoparticles that gained great interest for drug delivery. EVs have numerous advantages compared to synthetic nanoparticles, such as ideal biocompatibility, safety, ability to cross biological barriers and surface modification via genetic or chemical methods. On the other hand, the translation and the study of these carriers resulted difficult, mostly because of significant issues in up-scaling, synthesis and impractical methods of quality control. However, current manufacturing advances enable EV packaging with any therapeutic cargo, including DNA, RNA (for RNA vaccines and RNA therapeutics), proteins, peptides, RNA-protein complexes (including gene-editing complexes) and small molecules drugs. To date, an array of new and upgraded technologies have been introduced, substantially improving EV production, isolation, characterization and standardization. The used-to-be "gold standards" of EV manufacturing are now outdated, and the state-of-art requires extensive revision. This review re-evaluates the pipeline for EV industrial production and provides a critical overview of the modern technologies required for their synthesis and characterization.

In the past decade, extracellular vesicles (EVs) have emerged as key regulators of bone development, homeostasis and repair. EV-based therapies have the potential to circumnavigate key issues hindering the translation of cell-based therapies including functional tissue engraftment, uncontrolled differentiation and immunogenicity issues. Due to EVs' innate biocompatibility, low immunogenicity, and high physiochemical stability, these naturally-derived nanoparticles have garnered growing interest as potential acellular nanoscale therapeutics for a variety of diseases. Our increasing knowledge of the roles these cell-derived nanoparticles play, has made them an exciting focus in the development of novel pro-regenerative therapies for bone repair. Although these nano-sized vesicles have shown promise, their clinical translation is hindered due to several challenges in the EV supply chain, ultimately impacting therapeutic efficacy and yield. From the biochemical and biophysical stimulation of parental cells to the transition to scalable manufacture or maximising vesicles therapeutic response in vivo, a multitude of techniques have been employed to improve the clinical efficacy of EVs. This review explores state of the art bioengineering strategies to promote the therapeutic utility of vesicles beyond their native capacity, thus maximising the clinical potential of these pro-regenerative nanoscale therapeutics for bone repair.

Senescence of vascular smooth muscle cells (VSMCs) contributes to the formation of abdominal aortic aneurysm (AAA). Although mesenchymal stem cell exosomes (MSC-EXO) have been confirmed to restrict the development of AAA, their biological activity depends largely on the physiological state of the MSCs. This study aimed to compare the effects of adipose-derived MSC-EXO from healthy donors (HMEXO) and AAA patients (AMEXO) on senescence of VSMCs in AAA and explore the underlying mechanisms. An ApoE-/- mouse model of AAA was used to investigate the therapeutic effects of HMEXO, AMEXO or miR-19b-3p-AMEXO on AAA development. This in vitro model of AAA was established by treating VSMCs with Ang II (Angiotensin II). The senescence of VSMCs was determined by senescence-associated β-galactosidase (SA-β-gal) staining. The morphology of mitochondria in VSMCs was examined by MitoTracker staining. HMEXO exhibited superior capacity compared with AMEXO to inhibit VSMC senescence and attenuate AAA formation in Ang II-treated ApoE-/- mice. In vitro, both AMEXO and HMEXO inhibited Ang II-induced VSMC senescence via downregulation of mitochondrial fission. Notably, compared with HMEXO, the ability of AMEXO to inhibit VSMC senescence was significantly decreased. miRNA sequencing and the expression of miR-19b-3p was significantly decreased in AMEXO compared with HMEXO. Luciferase assay suggested that MST4 (Mammalian sterile-20-like kinase 4) is a potential target of miR-19b-3p. Mechanistically, miR-19b-3p in HMEXO ameliorated VSMC senescence by inhibiting mitochondrial fission via regulation of the MST4/ERK/Drp1 signaling pathway. Overexpression of miR-19b-3p in AMEXO improved their beneficial effect on AAA formation. Our study reveals that MSC-exosomal miR-19b-3p exerts protective effects against Ang II-induced AAA and VSMC senescence via regulation of the MST4/ERK/Drp1 pathway. The pathological state of AAA patients alters the miRNA components of AMEXO and impairs their therapeutic benefits.

The polarization of microglia plays an important role in the outcome of ischemic stroke (IS). In the aged population, senescent microglia show a predominant pro-inflammatory phenotype, which leads to worse outcomes in aged ischemic stroke compared to young ischemic stroke. Recent research demonstrated that inducible pluripotent stem cell-derived small extracellular vesicles (iPSC-sEVs) possess the significant anti-ageing ability. We hypothesized that iPSC-sEVs could alleviate microglia senescence to regulate microglia polarization in aged ischemic stroke. In this study, we showed that treatment with iPSC-sEVs significantly alleviated microglia senescence as indicated by the decreased senescence-associated proteins including P16, P21, P53, and γ-H2AX as well as the activity of SA-β-gal, and inhibited pro-inflammatory activation of microglia both in vivo and in vitro. Furthermore, iPSC-sEVs shifted microglia from pro-inflammatory phenotype to anti-inflammatory phenotype, which reduced the apoptosis of neurons, and improved the outcome of aged stroke mice. Mechanism studies showed that iPSC-sEVs reversed the loss of Rictor and downstream p-AKT (s473) in senescent microglia, which was involved in the senescence and pro-inflammatory phenotype regulation of microglia. Inhibition of Rictor abolished the iPSC-sEVs-afforded phosphorylation of AKT and alleviation of inflammation of senescent microglia. Proteomics results indicated that iPSC-sEVs carried transforming growth factor-β1 (TGF-β1) to upregulate Rictor and p-AKT in senescent microglia, which could be hindered by blocking TGF-β1. Taken together, our work demonstrates iPSC-sEVs reverse the senescent characteristic of microglia in aged brains and therefore improve the outcome after stroke, at least, via delivering TGF-β1 to upregulate Rictor and p-AKT. Our data suggest that iPSC-sEVs might be a novelty therapeutic method for aged ischemic stroke and other diseases involving senescent microglia.