Stem Cell–Derived Extracellular Vesicle Therapy in Ischemic Brain Injuries

Article information

J Stroke. 2025;27(3):313-328

Publication date (electronic) : 2025 September 29

doi : https://doi.org/10.5853/jos.2025.01690

Chunfang Qiu ¹, Abel Lindley ¹, Xiaofeng Jia^,¹^,²^,³

¹Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, MD, USA

²Department of Orthopedics, University of Maryland School of Medicine, Baltimore, MD, USA

³Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, USA

Correspondence: Xiaofeng Jia Department of Neurosurgery, University of Maryland School of Medicine, 10 South Pine Street, MSTF 823, Baltimore, MD, 21201, USA Tel: +1-410-706-5026 E-mail: XJia@som.umaryland.edu

Received 2025 April 11; Revised 2025 June 24; Accepted 2025 July 10.

Abstract

Ischemic brain injury (IBI), including stroke and cardiac arrest-induced global cerebral injury, presents a significant clinical challenge due to its high morbidity and incidence of neurological deficits. Currently, effective strategies for neurological repair remain limited. Extracellular vesicles (EVs) are a diverse group of cell-derived, lipid-bound nanoparticles that encapsulate RNAs, proteins, lipids, metabolites, growth factors, and cytokines. EVs play an essential role in intercellular communication and are involved in various physiological and pathological processes. Stem cell–derived EVs (SC-EVs) have been studied in the context of IBI, demonstrating regenerative and angiogenic effects that resemble those of their parent stem cells, holding promise for improved cell-free treatment of IBI. This review provides comprehensive insights into the therapeutic application of SC-EVs in IBI, including an SC-EV source comparison with their distinct advantages and limitations, and dissects the multifaceted mechanisms of SC-EVs including immunomodulation, neurogenesis, mitochondrial transfer, and myelin repair. Furthermore, it highlights recent advances in engineering SC-EV cargo and surfaces for enhanced targeting and efficacy in IBI treatment. It also emphasizes strategies to improve the reproducibility of in vivo studies through standardized protocols and bridges the gap between preclinical findings and early clinical trials. Finally, the review critically addresses ethical challenges and equity considerations, providing a roadmap for the responsible translation of SC-EV therapies into clinical practice.

Keywords: Ischemic brain injury; Stroke; Cardiac arrest; Extracellular vesicles; Stem cells

Introduction

Ischemic brain injury (IBI) arises from decreased cerebral blood flow and most commonly presents as ischemic stroke or cardiac arrest (CA)-induced cerebral injury, corresponding to focal and global IBI, respectively. Stroke remains the world’s second leading cause of death and the third leading cause of disability [1]. Similarly, cerebral hypoxia is the primary contributor to mortality and morbidity in patients who survive the initial CA event [2,3]. CA-related brain injury is also a leading cause of death and permanent neurological impairment worldwide, with substantial societal and healthcare burdens for survivors [4]. Despite advances in acute resuscitation, stroke care, and neurocritical medicine, no clinically effective treatments are available that mitigate the progression of IBI or promote significant neurological recovery.

Stem cell (SC) therapy has emerged as a promising therapeutic approach for treating IBI, showing neuroprotective, neurogenic, and functional benefits in preclinical IBI models [5-7]. However, their clinical translation is limited by immune rejection, potential tumorigenicity, poor delivery efficiency, and regulatory hurdles [8,9]. Growing evidence suggest these benefits are mediated largely by the paracrine actions of extracellular vesicles (EVs) [10-12], membrane-enclosed particles that transport bioactive cargo, including RNAs, miRNAs, proteins, lipids, and growth factors, to injured tissue [10,13,14]. Beyond their capacity for extensive expansion and diverse differentiation, stem cell–derived EVs (SC-EVs) have shown remarkable potential in the treatment of IBI due to their ability to cross the blood-brain barrier (BBB) and deliver therapeutic effects within the brain [14,15]. Furthermore, recent advances have focused on engineering SC-EVs to enhance targeting precision and biological efficacy, positioning EVs as a promising and more clinically viable alternative to cell-based therapies in ischemic contexts. However, key challenges remain to be addressed before EVs can be translated clinically, including their heterogeneous composition that hinders reproducibility across studies, limiting broader application.

In this review, we provide comprehensive insights into the therapeutic application of SC-EVs for focal and global IBI and discuss key pathophysiological features, such as immune cell infiltration and oxidative stress [16], shared by both IBI types that are studied in SC-EV preclinical studies. We offer a comparative analysis of multiple SC-EV sources with their distinct advantages and limitations, and dissects the multifaceted mechanisms including modulation of inflammation and apoptosis, promotion of neurogenesis and synaptogenesis, enhancement of angiogenesis and vascular remodeling, transfer of functional RNAs and mitochondria, and induction of oligodendrogenesis and myelin repair. We also highlight recent innovations in SC-EV cargo loading and surface engineering, specifically tailored to enhance targeting and efficacy in the ischemic brain. In addition, we propose practical strategies to improve the reproducibility of SC-EV therapies in preclinical studies through standardized experimental parameters such as dosage, timing, and delivery routes. A particularly important aspect of this review is our focused discussion on quality control and standardization strategies in SC-EV research. Finally, we bridge preclinical findings with emerging clinical data, while critically examining ethical and translational challenges, to support the responsible advancement of SC-EV-based therapies for IBI.

Biogenesis of SC-EVs

EVs are cell-derived, bilayer-enclosed membrane structures containing RNAs, miRNAs, proteins, lipids, metabolites, growth factors, and cytokines, acting as intercellular transporters among cells [10,13]. They are released by all cell types and can be broadly categorized into three subtypes based on their biogenesis and size: exosomes (30–150 nm), microvesicles (100–1,000 nm), and apoptotic bodies (1,000–5,000 nm). Exosomes are spheroid-shaped vesicles formed through the endosomal pathway, where membrane invagination forms early endosomes that internalize extracellular components and membrane proteins [17]. Microvesicles are irregular-shaped vesicles released by direct outward budding and fission of the plasma membrane, independent of the endosomal and apoptotic pathways [18]. Apoptotic bodies are irregular-shaped vesicles released by cells undergoing programmed cell death [19,20].

Cell types of SC-EVs and their roles in IBI

There are three main types of SC-EVs used for therapeutic purposes: embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (including mesenchymal, neural, and adipose stem cells). Each type of SC-EV offers unique therapeutic potential, with different mechanisms of action, benefits, and adverse effects in the treatment of IBI.

Embryonic stem cell–derived EVs

Embryonic stem cell–derived EVs (ESC-EVs) inherit the pluripotent nature of their parent ESCs, giving them broad paracrine effects that are highly relevant to both focal and global IBI [21]. In a rodent middle cerebral artery occlusion (MCAO) stroke model, intravenous (IV) administration of 1×10⁹ particles of ESC-EVs was shown to significantly reduce infarct volume, neuronal death, leukocyte infiltration, and tissue loss while improving long-term neurological function [21]. ESC-EVs can promote neuroprotection by maintaining the stemness and pluripotency of ESCs, preventing premature differentiation, and supporting the pluripotent state in vitro. In post-stroke treatment with ESC-EVs, it was shown that they expand regulatory T-cells (Tregs) and exert anti-inflammatory effects, thereby preserving their pluripotency, alleviating neuronal death, and retaining their capacity to generate chimeric mice [22].

However, the use of ESCs raises significant ethical concerns, primarily because their derivation involves the destruction of human embryos. Similarly, ESC-EVs presents various safety concerns, including the potential risk of tumor formation or uncontrolled cell growth, due to their propensity to promote cell proliferation [23,24]. Additionally, compared to other types of SC-EVs, ESC-EVs may elicit more pronounced immune responses due to their distinct molecular composition, although direct evidence remains limited [25]. ESCs are also known for their high telomerase activity and risk of teratoma formation, all of which may be reflected in their EVs [22,24]. These risks highlight the need for strategies to minimize immunogenicity and tumorigenicity in preclinical and clinical studies of ESC-EVs. Recent research on ESC-EVs is focused on understanding their cargo composition and their role in various biological processes, such as aging [26]. Advances in modifying ESC-EVs to improve targeting and address ethical concerns could broaden their acceptance in therapeutic applications.

Induced pluripotent stem cell–derived EVs

Induced pluripotent stem cell–derived EVs (iPSC-EVs) are reprogrammed somatic cells, such as fibroblasts or blood cells, converted back into a pluripotent state using specific transcription factors. Like ESCs, iPSCs can differentiate into nearly any cell type, including neurons, astrocytes, endothelial cells, and oligodendrocytes, all of which are essential for brain repair and functional recovery in IBI contexts. Since iPSCs can be generated from reprogrammed adult tissue, they offer a versatile and more ethically acceptable source for EVs [27-29], relative to ESCs. Recent preclinical studies have demonstrated the efficacy of iPSC-EVs in IBI. In MCAO mouse models, iPSC-EVs, when combined with electroacupuncture treatments, were shown to enhance neurological recovery, reduce neuronal damage, and improve overall functional outcomes [27]. Another study demonstrated that iPSC-EVs can reverse age-related BBB dysfunction and microglial senescence, thereby improving stroke outcomes in the MCAO model [29,30]. Given these mechanistic similarities, iPSC-EVs, like ESC-EVs, represent a promising strategy for newfound advances in the treatment of global cerebral ischemia. Nevertheless, before iPSC-EVs can translate to clinical intervention, it is essential to thoroughly assess any residual pluripotency-related tumorigenicity and immunogenicity risks.

Adult stem cell–derived EVs

Among the adult stem cells, mesenchymal stem cells (MSCs) and neural stem cells (NSCs) are the primary sources of EVs used for treating IBI.

Mesenchymal stem cell–derived EVs

Mesenchymal stem cell–derived EVs (MSC-EVs) are a type of adult stem cell and the most widely used source of SC-EVs in IBI research due to their ease of isolation, immunomodulatory properties, and established clinical safety. MSCs can be derived from various tissues, including adipose-derived MSCs (AD-MSCs or adipose-derived stem cell [ADSCs]) [31], known for their abundance and ease of isolation; umbilical cord–derived MSCs (UC-MSCs) [32], known for their high proliferative and immunomodulatory capacity; bone marrow–derived MSCs (BM-MSCs) [33], the most extensively studied; and dental pulp–derived MSCs (DP-MSCs) [34], recognized for their broad differentiation potential.

Each source of MSCs contributes uniquely to the regenerative potential of MSC-EVs in treating focal and global IBI. A single intranasal dose of human AD-MSC-EVs (200 μg/kg) administered 24 hours after ischemic stroke in rats significantly reduced infarct volume, improved BBB integrity, stabilized cerebral vasculature in the peri-infarct zone, and enhanced long-term motor and behavioral outcomes [35]. In a transient four-vessel occlusion rat model, human AD-MSC-EVs administered intranasally (3×10⁸ particles) for 7 days post-injury promoted long-term neuronal survival, and treated animals showed sustained cognitive improvements up to 28 days after global hypoxia [36]. Biodistribution analysis showed rapid EV accumulation in the olfactory bulb within 1 hour of AD-MSC-EV administration, where the EVs co-localized with microglia and significantly reduced expression of pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) [36].

UC-MSC-EVs have similarly been shown to cross the BBB and exert these beneficial effects, but also directly repair BBB disruption and significantly mitigate hemorrhagic transformation following thrombolysis with tissue plasminogen activator in ischemic stroke [37]. In a neonatal ischemic brain damage model, BM-MSC-EVs enriched with microRNA-410 (miR-410) were taken up by injured cortical neurons, resulting in reduced apoptosis and increased neuronal survival. Specifically, miR-410 targeted histone deacetylase 4 (HDAC4) to suppress wingless-related integration site (Wnt)/β-catenin signaling, a pathway associated with neuronal loss [38]. In a separate study, MSC-EVs loaded with miR-93 exerted similar results on the HDAC4/B-cell lymphoma 2 (Bcl-2) axis, significantly reducing infarct volume and pathological changes in hippocampal tissue [39]. Another research group also highlighted the role of miR-93, delivered by BM-MSC-EVs in a neonatal mouse model, to alleviate IBI through downregulation of the jumonji domain–containing protein 3 (JMJD3)/tumor protein p53 (p53)/kruppel-like factor 2 (KLF2) axis [40]. These findings highlight the broad and diverse array of MSC-EVs, each with distinct molecular cargo, regenerative profiles, and mechanisms of action, positioning MSC-EVs as a key platform for advancing cell-free therapies in IBI.

Neural stem cell–derived EVs

Neural stem cell–derived EVs (NSC-EVs) are another commonly used adult stem cell, which are found exclusively in the nervous system, capable of differentiating into neurons, astrocytes, and oligodendrocytes. The primary sources of NSCs are fetal NSCs, which produce EVs rich in pro-neurogenic factors, though their use faces ethical limitations.

In an MCAO rat model, NSCs modified to overexpress tumor susceptibility gene 101 released more NSC-EVs that were neuroprotective against stroke by reducing infarct volume, increasing anti-inflammatory effects, and enhancing levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor [41]. In another MCAO rat model, human NSC-EVs activated the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) pathway to directly promote neuronal survival, axonal growth, and synaptic plasticity [42]. In neonatal mice subjected to carotid ligation followed by hypoxia to induce hypoxic-ischemic brain injury (HIBI) analogous to CA-IBI, intranasal administration of NSC-EVs was shown to significantly decrease infarct size and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)+ apoptotic cells [43]. Collectively, these studies support NSC-EVs as an emerging and potent therapy option for brain injuries following stroke and CA.

Mechanisms of SC-EVs in the treatment of IBI

SC-EVs share common therapeutic mechanisms across both in vivo and in vitro models of IBI, though their effects vary largely due to differences in cargo composition. Understanding these similarities and differences is crucial for optimizing SC-EV therapies for specific IBI contexts (Figure 1).

Figure 1.

Function and mechanisms of stem cell–derived EVs (SC-EVs) in ischemic brain injury. OPCs, oligodendrocyte progenitor cells; BBB, blood–brain barrier; ROS, reactive oxygen species; miRNA, micro-RNAs.

Modulation of inflammation and apoptosis

SC-EVs exert neuroprotective effects in IBI primarily by modulating neuroinflammation and inhibiting neuronal apoptosis, two central mechanisms underlying their therapeutic effects. ESC-EVs were shown to promote neurological recovery after ischemic stroke by reducing leukocyte infiltration, inflammatory cytokines, neuronal death, and infarct volume while also increasing Treg levels; these neuroinflammatory effects are largely mediated by Tregs through the transforming growth factor beta (TGF-β)/small mothers against decapentaplegic (Smad) signaling pathway [21]. iPSC-EVs have also been associated with upregulating anti-apoptotic cytokines (IL-10, IL-17) to mitigate neuronal death, expand Tregs, and suppress pro-inflammatory T helper 1 (Th1) and T helper 17 (Th17) responses [27]. Notably, iPSC-EVs were observed to have synergistic potential with electroacupuncture that reduced neuronal apoptosis, dampened helper T-cell responses, and promoted Treg activity, suggesting a promising multimodal therapeutic strategy for IBI [44]. MSC-EVs also exhibit strong anti-inflammatory and neuroprotective effects through the delivery of bioactive molecules such as miR-21-5p [45], miR-124 [46], miR-223-3p [47], and TGF-β, which inhibit pro-inflammatory cytokine release and promote the reparative M2 phenotype shift of microglia [48]; this starkly contrasts the neurotoxic M1 microglia phenotype, which secretes pro-inflammatory cytokines and reactive oxygen species (ROS) [49]. MSC-EVs can also deliver antioxidants and anti-apoptotic molecules—including superoxide dismutase, catalase, miR-21-5p [50], and miR-133b [12]—that reduce oxidative stress by inhibiting caspase-3, activating the PI3K/Akt and Bcl-2 pathways [42], and limiting ROS accumulation [51]. These EVs have also been linked to reducing the infiltration of ED1+ macrophages expressing CD68 and Iba1+ microglial accumulation around infarcted brain regions [52]. ADSC-EVs enriched with miR-126 were also found to attenuate neuroinflammation and promote recovery in rodent MCAO models through microglial suppression [53]. Collectively, these bioactive molecules reduce the secretion of inflammatory cytokines (IL-1β, IL-6, TNF-α), increase the secretion of anti-inflammatory cytokines (IL-10), and suppress microglial-mediated neuroinflammation in IBI contexts [46].

Promotion of neurogenesis and synaptogenesis

SC-EVs enhance recovery after ischemic injury by promoting neuron regeneration and synaptic remodeling. iPSC-derived neural progenitor cell EVs (iPSC-NPC-EVs) have been shown to increase the secretion of BDNF, fibroblast growth factor, and miR-9 [54], which function to guide neural progenitors towards infarct sites, stimulate neurite outgrowth, and improve synaptic communication. MSC-EVs similarly support synaptic repair by delivering neurogenic molecules like synapsin I, growth-associated protein 43 (GAP-43), and miR-132 that enhance synaptic formation and remodeling [55-57]. In a rat MCAO model, BM-MSC-EVs were found to activate JNK1/c-Jun signaling pathways that regulate neuronal apoptosis and synaptic-axonal remodeling, ultimately through the differentiation of NSCs [58]. Intracerebroventricular injection of 200 μL of BM-MSC-EVs (obtained from plates that yield 5 mg EV protein per 1 mL serum) in transient global ischemia mice models, where the common carotid arteries were occluded, restored synaptic transmission and neural plasticity, with improved performance in the Morris water maze, possibly due to preserved long-term potentiation through the inhibited expression of cyclooxygenase-2 in the hippocampus [59].

Enhancement of angiogenesis and vascular remodeling

Restoring blood supply to the ischemic brain is crucial for tissue repair and functional recovery. SC-EVs promote angiogenesis and vascular remodeling by delivery pro-angiogenic factors that stimulate endothelial cell proliferation [45,60,61], strengthening the brain’s ability to repair itself and improving overall recovery. ADSC-EVs carrying miR-181b-5p and miR-212-5p were shown to increase endothelial cell mobility and overall angiogenesis following oxygen-glucose deprivation (OGD) in an MCAO rat model [62]. iPSC-EVs also support angiogenesis by inhibiting signal transducer and activator of transcription-3 (STAT-3)-dependent autophagy, while human-iPSC-MSC-EVs, BM-MSC-EVs, and iPSC-EVs deliver vascular endothelial growth factor (VEGF) [63], miR-126 [60], and miR-21-5p [45], respectively, to drive vascular growth. In addition to promoting angiogenesis, iPSC-EVs improve BBB integrity in aged mice [29], while EV-delivered miRNAs such as miR-125b-5p reinforce endothelial tight junctions and reduce permeability [37]. Notably, the synergistic effects of Houshiheisan, a traditional Chinese medicine, and endothelial progenitor cell–derived EVs (EPC-EVs) in cerebral ischemia were potentiated significantly compared to equivalent doses of unmodified EP-CEVs. Specifically, MCAO and OGD rats had improved cortical perfusion and cortical microvessel density, likely through the actions of miR-126 on the PI3K subunit 2 (PIK3R2)/PI3K/Akt pathway [64].

Transfer of functional RNAs and mitochondria

Emerging studies suggest that SC-EVs can transfer functional RNAs and mitochondria to injured cells, significantly enhancing their metabolic function and promoting survival under ischemic conditions. The delivery of mitochondria within microvesicles to damaged brain endothelial cells (BEC) increased ATP levels by 100 to 200-fold (relative to untreated cells) in an OGD model in vitro [65]. In a mouse MCAO model, mitochondria-containing EVs derived from mouse BECs improved therapeutic efficacy for ischemic stroke [66]. This mitochondrial transfer not only helps restore cellular energy levels but also supports the overall recovery of dysfunctional neuronal cells. In addition to mitochondrial transfer, SC-EVs also facilitate miRNA-mediated gene regulation by delivering various miRNAs that regulate key genes involved in IBI. One study demonstrated that miRNA-126 genetic overexpression in modified ADSC-EVs promoted functional recovery in an MCAO rat model by improving neurogenesis and suppressing microglia activation [53]. Another study confirmed that miR-25, enriched in AD-MSC-EVs, significantly inhibited autophagic flux through the p53-Bcl-2/adenovirus E1B 19kDa-interacting protein 3 (BNIP3) signaling pathway, reduced infarct size, and improved neurological recovery in an MCAO mouse model [31]. Other important functional miRNAs found in SC-EVs for IBI treatment include miR-21-5p, which targets pro-apoptotic genes [45], and miR-126, which regulates genes involved in angiogenesis [60]. By modulating gene expression, SC-EVs enhance the brain’s intrinsic ability to repair itself, thus playing a crucial role in promoting recovery following ischemic injury.

Induction of oligodendrogenesis and myelin repair

Damage to white matter, particularly the loss of myelin, is a significant consequence of IBI. SC-EVs play a crucial role in supporting the regeneration of oligodendrocytes and the repair of myelin sheaths, both of which are essential for restoring proper nerve conduction after IBI. In an MCAO mouse model, NSC-EVs which contained miR-128-3p promoted oligodendrocyte precursor cells (OPCs) differentiation and remyelination by inhibiting fibrinogen-mediated bone morphogenetic protein (BMP) signaling, offering a potential therapeutic strategy for ischemic stroke [67]. Specifically, SC-EVs can carry factors like insulin-like growth factor (IGF-1) and miR-128-3p to increase OPC differentiation into mature oligodendrocytes; this is vital for the re-myelination process, enabling axons to effectively conduct nerve impulses, highlighting the potential of SC-EVs as a multifaceted therapeutic strategy for restoring neural functional recovery in the postischemic brain [67,68].

Advances in drug loading and manufacturing of SC-EVs

SC-EVs are emerging as powerful therapeutic agents due to their ability to deliver biologically active molecules to target tissues. However, clinical translation is limited by challenges such as low targeting efficiency and insufficient cargo loading [69]. As such, recent improvements focus on three key areas: (1) optimizing the cargo carried by SC-EVs; (2) engineering their surfaces to enhance targeting, stability, and therapeutic efficacy; and (3) developing scalable manufacturing techniques.

Advances in cargo engineering

Cargo engineering focuses on enhancing the therapeutic potential of SC-EVs by loading them with various biologically active molecules such as proteins, RNAs, or small-molecule drugs. Recent advancements aim to optimize the encapsulation processes, increase the loading capacity of these vesicles, and improve their delivery to target cells [70,71]. RNA-based cargo loading is a pivotal strategy, incorporating therapeutic RNA molecules like miRNAs, small interfering RNAs (siRNAs), and messenger RNAs (mRNAs). BM-MSC-EVs loaded with miR-93 or miR-410 can target nuclear proteins to suppress Bcl-2 family proteins, which are a key regulator of cellular apoptosis and neuronal loss [38,39]. Furthermore, modifying MSC-EVs via IL-6 stimulation at 1 ng/mL was shown to increase miR-455-3p expression in their exosomes to promote cell proliferation [72].

EVs synergize well with various chemical and biomedical interventions that have shown promise in improving their efficiency. Beyond the previously mentioned Houshiheisan-modified EVs [64], resveratrol has also been used to boost the protective effects of NSC-EVs from ROS by increasing the expression of mitochondrial biogenesis genes including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear respiratory factor 1 (NRF1) [73].

Rhein has shown promise in having dose-dependent neuroprotective effects on cerebral ischemia/reperfusion, OGD, and MCAO injured rats. This was mediated through the inhibition of nuclear factor erythroid 2-related factor 2 (NRF2) in the NRF2/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) axis, which ultimately reduced oxidative stress and harmful ferroptosis-related protein expression, laying the framework for future iron-based modifications for SC-EV therapies in focal and global IBI [74].

Curcumin-modified EVs in IBI contexts have also been shown to inhibit ROS-mediated apoptosis in rats, where its innate antioxidant properties can restore the mitochondria membrane potential and reduce cytochrome c release [75].

Techniques such as electroporation facilitate efficient loading of neuroprotective miRNAs, while siRNAs can target inflammatory or apoptotic pathways in ischemic neurons [76]. Additionally, mRNAs encoding protective proteins like BDNF can be packaged into EVs via lipid nanoparticle-assisted transfection, allowing for the targeted delivery of functional proteins [77]. In addition to RNA, sonication and extrusion techniques can temporarily disrupt SC-EV membranes, enabling the encapsulation of antioxidants and anti-inflammatory drugs [78,79]. Furthermore, surface-active proteins, including BDNF and IGF-1, can be incorporated in EVs to augment their neuroprotective properties. This can be achieved using fusion proteins with EV-targeting domains to ensure precise localization [77,80]. Other strategies are also exploring the delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) components via SC-EVs for gene editing, targeting genes that exacerbate brain damage during ischemia. This approach involves loading EVs with Cas9 protein and guide RNAs through transfection or co-culture with CRISPR-engineered stem cells, offering a promising avenue for targeted therapeutic interventions [81]. AD-MSCEVs constructed to overexpress CD47 were found to escape macrophage uptake, thereby increasing the circulation time of CD47-overexpressing EVs. Subsequently, this modification reduces the immune clearance of EVs and improves the overall drug delivery [82].

Surface engineering techniques

Surface engineering is another essential technique for enhancing the specificity and biodistribution of SC-EVs in the treatment of IBI. Techniques such as targeting ligand attachment enable SC-EVs to recognize specific receptors on the BBB and injured brain cells, facilitating more efficient delivery. Furthermore, peptide-based ligands, such as rabies virus glycoprotein and T7 peptides, can be conjugated to SC-EVs, enhancing their ability to cross the BBB. For example, surface-modified SC-EVs conjugated with anti-GAP43 monoclonal antibodies effectively targeted damaged neurons, enhancing quercetin delivery, reducing oxidative stress via NRF2/heme oxygenase-1 (HO-1) activation, and improving neuroprotection against cerebral ischemia and reperfusion injury [69].

Hybrid EVs, which combine SC-EVs with nanoparticles, further improve therapeutic efficacy and targeting capabilities. Magnetic nanoparticles enable the precise delivery of SC-EVs to ischemic regions [83]. A study found that magnetic EVs, derived from iron oxide nanoparticles, can increase the expression of MSC-EVs’ endogenous growth factors to promote angiogenesis and reduce apoptosis in ischemic stroke. Importantly, this surface modification was able to improve the targeting of MSC-EVs to better populate ischemic brain regions, significantly reducing overall infarction volume and improving motor recovery [83]. Moreover, surface modifications like polyethylene glycosylation help improve the circulation time of SC-EVs and allow them to evade the immune system, increasing their effectiveness in delivering therapies to the ischemic brain [84]. A previous study explored a method for preparing phosphatidylserine-deficient small EV (sEV) (PS^(-)sEVs) subpopulations that circulate longer in the bloodstream, created by an enzymatic reaction with phosphatidylserine decarboxylase (PSD) [85]. The results of this study demonstrated that PSD treatment effectively depleted surface phosphatidylserine and exhibited a prolonged circulation half-life, increasing the yield of PS^(-)sEVs without altering their physical properties and suggesting the potential for enhanced therapeutic applications [85]. Hydrogen sulfide (H₂S) preconditioning of MSC-EVs has also shown substantial neuroprotective effects in global cerebral hypoxiaischemia in neonatal mice. Preconditioned MSC-EVs were taken up more readily by microglia and neurons of injured brain regions, delivering miR-7b-5p at significantly greater concentrations than unconditioned MSC-EVs. The molecular consequences were that H₂S preconditioning increased anti-inflammatory phenotypes of microglia and brain mononuclear phagocytes, decreasing the release of inflammatory cytokines and promoting long-term neurocognitive function [13].

Scalable manufacturing techniques

As SC-EVs advance toward clinical applications, scalable and reproducible manufacturing techniques are crucial for ensuring their quality, potency, and safety [86]. Recent innovations have focused on bioreactor systems that enable continuous culture and harvesting of SC-EVs, significantly improving yield [87]. Additionally, techniques like tangential flow filtration facilitate the efficient separation and concentration of EVs from large culture volumes, while 3D culture systems—which mimic the natural stem cell environment—produce EVs with enhanced therapeutic properties, especially for IBI [88]. Moreover, purification methods such as size exclusion chromatography and immunoaffinity capture are optimized to isolate high-purity SC-EVs without compromising their integrity. Throughout all of these manufacturing approaches, quality control measures are integral to maintaining the therapeutic efficacy of SC-EVs, and various techniques—such as nanoparticle tracking analysis (NTA), flow cytometry, and proteomic/RNA profiling—are employed to standardize EV size, concentration, and cargo content. These advancements in purification and quality control not only ensure batch-to-batch consistency but also enhance the overall effectiveness of SC-EV therapeutics in clinical settings.

Optimizing the efficiency and reproducibility of SC-EVs treatment in in vivo studies of IBI

Although numerous preclinical studies have explored the therapeutic effects of SC-EVs in various IBI models (Table 1), significant variability in experimental design, such as stem cell source, isolation methods, dosing, and delivery routes, continues to limit SC-EVs’ reproducibility. The Minimal Information for Studies of Extracellular Vesicles (MISEV2023) guidelines have provided foundational recommendations for EV isolation and characterization [89], but must be further refined to bring SC-EV therapy at the forefront of clinical translation.

Table 1.

Therapeutic strategies and mechanisms of SC-EVs in animal models of ischemic brain injury

Quality control of SC-EVs remains a major challenge that has recently been outlined via critical quality attributes (CQAs), such as vesicle size, purity, surface markers, and cargo, to minimize batch-to-batch variation. This includes validating EV identity via surface marker profiling (e.g., CD63, CD81, Alix), confirming purity through electron microscopy and NTA, and establishing functional potency assays to assess biological activity. Consistent EV production involves the use of well-characterized stem cell sources, such as BM-MSCs, UC-MSCs, or NSCs; widely-known cell culture and purification methods, including ultracentrifugation and size-exclusion chromatography; and robust characterization techniques (e.g., NTA, transmission electron microscopy, and western blotting) to ensure SC-EV purity and standardization in treating IBI.

Delivery optimization is also critical. While intranasal and IV routes are practical options, intracerebroventricular administration provides more precise targeting to ischemic brain regions. Advanced approaches, such as engineering EVs with brain-specific ligands or encapsulating them in hydrogels or nanoparticles, can further enhance targeting efficiency and therapeutic sustainability. Proof-of-concept studies centered on understanding the mechanisms behind engineered SC-EVs are also needed to understand the biochemical and molecular mechanisms behind their neuroprotective effects in human IBI.

To enhance SC-EVs’ reproducibility across preclinical and rising clinical studies, time-dependent intervention considerations [90] and dose-escalation studies must be considered. Other strategies, such as tailoring EV cargo by loading specific neuroprotective miRNAs [91] or proteins [92], may improve outcomes in neurogenesis and inflammation regulation. In vivo tracking techniques, such as fluorescent labeling and magnetic resonance imaging, help optimize the efficiency and reproducibility of SC-EV therapy for IBI by enabling precise monitoring of their biodistribution and targeting in real time. Rigorous assessments of neurobehavioral recovery, histopathological changes, and molecular markers ensure comprehensive evaluation and enhance the translational potential of SC-EVs for IBI, such as the Stroke Therapy Academic-Industry Roundtable (STAIR) and Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) guidelines. Integrating these elements alongside MISEV2023 and CQA frameworks will improve SC-EV comparability across studies and support their advancement toward clinical application.

Clinical investigation of SC-EVs in human IBI contexts

Recent clinical investigations across ischemic contexts have provided preliminary evidence supporting the safety and therapeutic promise of SC-EVs (Table 2). Notably, a pilot randomized clinical trial (RCT) including 10 patients undergoing decompressive craniectomy for malignant middle cerebral artery infarction showed that placenta-derived MSC (PMSC)-EVs were safe and associated with significantly greater improvements in consciousness, motor strength, speech, and daily functional outcomes, suggesting potential neurorestorative effects in severe ischemic stroke [93]. The intervention group (n=5) received a single dose of 1 mL allogeneic PMSC-EVs containing 1×10⁹ particles/mL intraoperatively via stereotactic injection into the ischemic penumbra region.

Table 2.

Clinical studies of EV therapy in IBI and related conditions

In the context of focal and global IBI and SC-EV therapy, there are six clinical studies that are either completed or in Phase I reporting. Across the three studies that have conclusive data, SC-EV treatments were reported to be safe and well-tolerated, with no serious adverse events or immune-related reactions. The remaining three clinical trials involve various SC-EV types and doses as direct interventions for IBI, such as 4×10⁹ and 1.6×10¹⁰ NSC-EV particles/kg. Nonetheless, larger, controlled trials are still needed to confirm long-term immunological safety, especially in repeated or systemic dosing scenarios of SC-EVs.

Challenges and prospects of SC-EVs in IBI

SC-EVs hold significant potential as therapeutic agents for IBI, but various challenges must be addressed before they can be widely implemented in clinical settings [94-96]. The therapeutic efficacy of SC-EVs still has not been fully validated in human applications, with variability in clinical outcomes reported in these studies. This variability primarily arises from the inherent heterogeneity of EV cargo, which is highly influenced by multiple variables, including the cell type, cell state, culture conditions, isolation techniques, and quantification methods. Similarly, the timing and frequency of EV administration present additional complexities, making it difficult to draw generalized conclusions or conduct meaningful cross-comparisons. Furthermore, the scalability and manufacturing of SC-EVs present challenges in producing sufficient quantities while maintaining their therapeutic potency. To overcome these hurdles, future research should focus on several critical areas. Developing standardized isolation and characterization techniques will help address the heterogeneity of SC-EVs, while improving delivery methods—such as surface modifications to enhance BBB penetration and responsive delivery systems—may facilitate more targeted treatment. Additionally, enhancing the therapeutic efficacy of SC-EVs through cargo engineering, including the loading of gene-editing systems and combinatorial therapies, may further increase their impact. Efforts to improve scalability and manufacturing, combined with well-designed clinical trials, are essential to transition SC-EV therapies from the bench to clinical applications. Moreover, combining SC-EVs with other therapeutic approaches may help overcome some of the limitations associated with EV therapy. For example, the combined effects of electroacupuncture and iPSC-EVs in MCAO mouse models demonstrated reduced infarct volume and exerted neuroprotective effects by modulating the IL-33/suppression of tumorigenicity 2 (ST2) axis [44]. This suggests that synergistic SC-EV regimens with other treatment modalities could become a transformative therapy for treating IBI.

Ethical challenges of SC-EVs in clinical practice

The clinical application of SC-EVs presents several ethical challenges that need to be addressed before such therapies can be widely implemented. One of the most contentious issues involves the use of ESCs, which require the destruction of human embryos for their extraction; as a direct result, this raises ethical questions about the balance between the potential therapeutic benefits and the moral considerations surrounding the beginning of life. Public policy discussions have largely focused on defining the latter to better address the limitations of using embryos for scientific purposes [97], and as advancements in this field progress, these debates will remain crucial in shaping the ethical frameworks for ESC-based therapies, ensuring that scientific progress remains aligned with societal values. However, opinions on embryo research vary widely: some consider all embryo research unacceptable, while others only support certain forms like infertility research. As such, a pillar of bioethical thought that must be held to the highest standards is obtaining informed consent with all needed regulation approval, including institutional review board (IRB) approvals, which ensures respect for individual autonomy and recognizes the diversity of perspectives on the moral implications of embryo use.

In addition to the ethical concerns surrounding the use of embryos, the clinical application of SC-EVs also raises important issues about safety, long-term effects, and equitable accessibility. A major obstacle to their clinical translation lies in the considerable variability in SC-EV quality, purity, sourcing, and production methods, all of which complicate efforts to standardize therapies and ensure reproducible therapeutic outcomes. Notably, inconsistencies in immunomodulatory effects, such as conflicting data on T-lymphocyte modulation, have been linked to divergent EV isolation and processing protocols [98,99]. Moreover, as the costs and technological expertise required for SC-EV therapies increase, there is growing concern that these therapies may remain accessible only to privileged populations, thereby widening existing healthcare disparities [100]. Another critical issue is genomic inequality, where the reliance on limited or one-person trials may lead to inequitable outcomes and hinder the generalizability of SC-EV therapies across diverse populations [101,102]. To overcome these challenges, robust and transparent regulatory frameworks are urgently needed. These should not only enforce rigorous safety and quality standards but also promote the development of inclusive clinical trials and equitable access pathways, thereby ensuring that the benefits of SC-EV therapies can be safely and fairly delivered across diverse patient populations.

Conclusions

In summary, SC-EVs present a promising therapeutic approach for treating IBI, leveraging their ability to deliver bioactive molecules that promote neuroprotection, neurogenesis, and angiogenesis. However, several significant challenges must be addressed to facilitate their successful clinical application, including heterogeneity of SC-EV cargos, limited delivery across BBB, potential immunogenicity, and lack of reliable large-scale manufacturing platforms for SC-EVs. This requires future efforts in standardization in isolation and characterization, delivery optimization, cargo engineering, and rigorous clinical validation. Additionally, conducting well-designed clinical trials will be crucial for validating the safety and efficacy of SC-EV therapies in human patients. By overcoming these multifaceted challenges, SC-EVs have the potential to revolutionize the treatment landscape for patients suffering from IBI, offering hope for more effective therapeutic outcomes and improved quality of life.

Notes

Funding statement

This work was partially supported by 2024-MSCRFD-6401 from the Maryland Stem Cell Research Fund, USA and R01NS125232 and R01NS110387 from the National Institute of Neurological Disorders and Stroke, USA (all to Xiaofeng Jia).

Conflicts of interest

The authors have no financial conflicts of interest.

Author contribution

Conceptualization: XJ. Study design: XJ. Methodology: XJ. Data collection: CQ, AL. Investigation: CQ. Writing—original draft: CQ. Writing—review & editing: CQ, AL, XJ. Funding acquisition: XJ. Approval of final manuscript: CQ, AL, XJ.

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Type(s) of SC-EV	Animal model	Disease model	Dose amount	Route	Administration	Dose schedule	Mechanism	Reference
ESC-sEV	Mouse	MCAO	10⁹ Particles	IV	2 h post-MCAO	TID for 3 days	Modulates neuroinflammation and immune responses	[21]
iPSC-sEV	Mouse	MCAO	10⁹ Particles	Tail IV	24 h post-stroke	Single dose	Rejuvenates senescent BBB	[29]
iPSC-iMSC-sEV	Rat	MCAO	10¹¹ Particles	Tail IV	24 h post-MCAO	Single dose	↑Angiogenesis	[28]
hiPSC-MSC-EV	Mouse	MCAO	100 μg	IV	Days 1, 3, and 5 post-MCAO	Once on each day	↑Angiogenesis, ↑Cell proliferation, ↓Apoptosis, Modulates cellular morphology	[63]
MSC-EV	Rat	MCAO	200 μL	Tail IV	24 h & 15 days post-MCAO	Repeated	↓Microglial M1 polarization-mediated inflammation	[47]
MSC-EV	Rat	MCAO	3×10¹¹ Particles	IV	Immediately post-MCAO	Single dose	↑Microglia Polarization (M2 phenotype)	[103]
MSC-EV	Rat	MCAO	200 μg	IV	Immediately after reperfusion	Single dose	↑Blood vessel density, ↑Macrophage polarization (M2 phenotype)	[83]
MSC-EV	Ovine	Neonatal HIBI	2×10⁷ cell equivalents	IV	1 h and 4 days following HIE	Repeated	↑BBB integrity, prevents HIBI	[104]
MSC-EV	Ovine	Neonatal HIBI	2.0×10⁷ cell equivalents	IV	1 h and 4 days following HIE	Repeated	Preserves cortical function and baroreflex sensitivity; partially protects against hypomyelination; no protection against apoptosis or neuroinflammation	[105]
MSC-sEV	Mouse	MCAO	Equivalent to 2×10⁶ MSCs	Tail IV	Immediately or 6 h post-MCAO	Single dose	↓Neuronal injury, ↓Inflammation, ↓Leukocyte infiltrate	[106]
hWJ-MSC-EV	Rat	HIBI	50 mg/kg	IN	2 h post-HIBI	Single dose	↓Microglia-mediated neuroinflammation	[107]
rAD-MSC-EV	Rat	MCAO	100μg/kg/day	ICV	Prior to MCAO	Daily for 3 days	↓Brain injury, ↑Neuron viability, ↓Apoptosis	[108]
hAD-MSC-EV	Rat	MCAO	200 μg/kg	IN	24 h post-MCAO	Single dose	↑BBB vascularization	[35]
hAD-MSC-EV	Rat	HIBI	3×10⁸ Particles	IN	30 min post-HIBI	Daily for up to 7 days	↑Cognitive function, ↓Apoptosis, ↑Neuronal survival in hippocampus, ↓Inflammatory cytokines	[36]
UC-MSC-EV	Rat	MCAO	80 μg	Tail IV	Start of reperfusion	Daily for 2 days	↑Vascularization	[32]
hUC-MSC-EV	Mouse	MCAO	200 µg	Tail IV	2 h post-MCAO	Single dose	↓tPA-induced BBB disruption	[37]
hUSC-EV	Rat	MCAO	10¹¹ Particles	Tail IV	4 h post-MCAO	Single dose	↑Neurogenesis	[109]
BM-MSC-EV	Mouse	MCAO	100 µg	Tail IV	30 min post-MCAO	Single dose	↓Neuronal death induced by OGD	[110]
BM-MSC-EV^*	Rat	MCAO	100 μg	Tail IV	30 min post-MCAO	Single dose	↓BBB permeability	[111]
BM-MSC-EV	Rat	MCAO	200 μg	Tail IV	Start of reperfusion	Single dose	↓Pyroptosis	[112]
BM-MSC-EV	Rat	MCAO	10¹⁰ Particles	Tail IV	Immediately post-MCAO	Single dose	↓BBB permeability	[113]
BM-MSC-EV	Rat	MCAO	3×10¹¹ Particles	Tail IV	24 h post-stroke	Single dose	↓Apoptosis and ↓Synaptic-axonal remodeling	[58]
mBM-MSC-EV	Mouse	MCAO	100 μg	Tail IV	Immediately after reperfusion	Single dose	↓Infarct area, ↓Neuronal injury, ↓Apoptosis	[114]
rBM-MSC-EV	Mouse	MCAO	25–50 μg	Tail IV	24 h post-MCAO	Single dose	↑Angiogenesis	[45]
hBM-MSC-EV	Rat	MCAO	Low dose (2×10⁶ MSC equivalents/kg); High dose (2×10⁷ MSC equivalents/kg)	Tail IV	Days 1, 3, and 7 post-stroke	Once on each day	↓Macrophage infiltrates, ↑Angiogenesis, ↑Neurogenesis	[52]
hDP-MSC-EV	Mouse	MCAO	10 µg	Tail IV	2 h post-reperfusion	Single dose	↓Inflammation	[34]
NPC-EV	Mouse	MCAO	2.5–3.7×10¹⁰ Particles	Tail IV	12 h post-reperfusion	Single dose	↓Inflammation	[115]
iNPC-EV	Mouse	MCAO	150 μg	IV	24 h post-MCAO & 2 d intervals on days 1–5 post-stroke	Once on each day	↑Sensorimotor function recovery, ↑cognitive function, ↑NPC proliferation, ↓Inflammation, ↓Apoptosis	[116]
mNPC-EV	Mouse	MCAO	Low (10 μg, from 2×10⁵ NPCs); Medium (100 μg, from 2×10⁶ NPCs); and High (1,000 μg, from 2×10⁷ NPCs)	Femoral IV	Days 1, 3, and 5 post-stroke	Once on each day	↑Long-term neuroprotection, ↑Cell proliferation, ↑Neuroregeneration, ↑Axonal plasticity, ↑Immunosuppression	[117]
NSC-EV	Rat	MCAO	30 μg	ICV	2 h post-stroke	Single dose	↓Microglial density, ↓Apoptosis	[118]
NSC-EV	Rat	MCAO	100 μg	Tail IV	Immediately post-MCAO	Daily for 28 days	↑NSC proliferation, ↑NSC differentiation	[119]
rNSC-EV	Rat	MCAO	100 µg	Tail IV	24 h post-reperfusion	Single dose	↓Pyroptosis	[120]
hNSC-EV	Rat	MCAO	4×10⁹ Particles	ICV	24 h post-stroke	Single dose	↑Cell proliferation, ↓Apoptosis	[121]
hiNSC-EV	Mouse	MCAO	10 μg	ICV	Immediately post-MCAO	Single dose	↑NSC differentiation, ↓Oxidative stress, ↓Inflammation, ↓Glial scar formation	[122]

Condition	Location	EV description	Administration	Results / Effectiveness	Adverse effects	Reference or NCT #
Acute ischemic stroke	Tehran, Iran	356 µg/mL of allogenic placenta-MSC-EV	One intraparenchymal injection	↑Consciousness, ↑Motor function, ↑Speech, ↑Activity	No reported AEs	NCT03384433 (2019–2021) [93]
Stroke, including acute ischemic	Florence, Italy; Milano, Italy	N/A	N/A	Surface plasmon resonance imaging biosensor can characterize different EVs as stroke biomarkers	No reported AEs	NCT05370105 (2018–2022)
Acute ischemic stroke	Seoul, Republic of Korea	4.8×10¹⁰, 9.6×10¹⁰, 19.2×10¹⁰ MSC-EV particles/kg	IV QD×5 d	Human trial on-going; in non-human primates: ↑neurological recovery	–	NCT06995625 (2025–Present) [123]
Acute ischemic stroke	Wuhan, China	4×10⁹, 8×10⁹, 1.6 ×10¹⁰ NouvSoma001 (hiNSC-EV) particles/kg	IV QD×7 d	Trial on-going	–	NCT06612710 (2025–Present)
Neonatal hypoxia-ischemia	Moscow, Russian Federation	12 million MSC-EV particles	IN QOD×5	Trial on-going	–	NCT05490173 (2022–Present)
Transient ischemic attack	Stockholm, Sweden	N/A	N/A	PS⁺TF⁺ platelet microvesicles are a marker of cerebral ischemia	No reported AEs	NCT05524506 (2007–2021)