Introduction

Regenerative medicine and oncology are vital fields in the fight against the global burden of chronic diseases and cancers. Regenerative medicine aims to repair or replace tissues and organs damaged by severe injuries or chronic disease, while oncology focuses on combating the complex pathophysiology of cancers. With the aging population and rising incidence of chronic diseases such as cardiovascular diseases, neurodegenerative diseases and osteoarthritis, alongside the continued prevalence of cancers, the demand for innovative treatment plans has intensified.1–4 Conventional therapeutic approaches such as pharmacological interventions, surgical transplantation, and chemotherapy can alleviate symptoms but often fail to restore tissue function, and frequently associated with adverse effects and high costs. For instance, organ transplantation faces limitations due to donor shortages and immune rejection risks,5 while chemotherapies and targeted therapies face challenges such as systemic toxicity, drug resistance, and tumor recurrence.6,7 These limitations emphasize the urgent need for therapies that can restore tissue integrity, regulate the disease microenvironment, and precisely target multifactorial pathology.

Mesenchymal stem cells (MSCs) are recognized as a cornerstone therapeutic agent in regenerative medicine due to their trilineage differentiation capacity, immunomodulatory properties, and paracrine signaling capabilities.8 Nevertheless, MSC-based therapies face critical translational challenges: poor post-transplantation survival rates, inefficient homing to injury sites,9 potential tumorigenic risks,10 and regulatory complexities.11 These challenges have prompted a shift in research focus towards the utilization of extracellular vesicles (EVs), which are nanoscale lipid bilayer particles secreted by MSCs, as a cell-free alternative.12 EVs are generated through distinct biogenesis pathways, such as the endosomal sorting complex required for transport (ESCRT)-dependent mechanism and ESCRT-independent processes that involve tetraspanin-rich microdomains, which decide their molecular composition and functional specificity.13 EVs inherit therapeutic cargo from parent cells, including proteins, nucleic acids, lipids, and metabolites, while avoiding risks associated with whole cell therapy. Their low immunogenicity, stability, and ability to traverse biological barriers position EVs as versatile vectors for both regenerative and oncological applications.14 It is important to note that while EVs are not devoid of challenges related to limited targeting efficiency, potential pro-tumorigenic effects in certain environments, and regulatory hurdles, they present distinct advantages: their nanoscale size offers more favorable biodistribution and penetration, and their synthetic flexibility makes them highly suitable for enhancing homing engineering strategies;15 their anucleate nature prevents them from proliferating, thus significantly reducing the risk of tumorigenesis;16 and they are subject to a different, although still evolving, regulatory pathway as biological products rather than live cells.

Human umbilical cord-derived MSCs-EVs (hucMSCs-EVs) are particularly promising because they can be obtained non-invasively from medical waste, proliferate rapidly, have low immunogenicity, and do not raise ethical issues.17 In regenerative medicine, hucMSCs-EVs demonstrate efficacy in different systems such as the nervous system, locomotor system, and respiratory system by modulating inflammation, promoting angiogenesis, and stimulating tissue repair.18–22 At the same time, their role in cancer therapy is being increasingly recognized: hucMSCs-EVs deliver tumor-suppressive miRNAs, reverse chemoresistance, and remodel tumor microenvironments (TME) in cancers such as gastric, prostate, and ovarian malignancies.23–27 The dual therapeutic strategies of regeneration and cancer therapy empower hucMSCs-EVs to serve as a “multi-treatment tool” for combating various disease.

Despite the promising therapeutic potential of hucMSCs-EVs, several critical unknowns remain. The precise mechanisms controlling their biodistribution, cellular uptake, and cargo sorting are not fully elucidated. Additionally, the heterogeneity of EVs populations due to variations in isolation methods and source conditions poses challenges for standardization. The long-term safety profile, including potential off-target effects and immunogenic responses, especially in immunocompromised patients, requires further investigation. Moreover, the influence of the parental cell’s physiological state on EVs functionality is poorly understood. Finally, while preclinical models demonstrate efficacy, the extension to human diseases is limited by species-specific differences and complex disease microenvironments. Addressing these gaps is essential for the rational design and clinical translation of hucMSCs-EVs-based therapies. We focus on the latest progress in hucMSCs-EVs for regenerative medicine and oncology, while also critically discussing the above challenges and future perspectives necessary to advance this booming field.

Mechanisms of EVs Biogenesis

EVs are mainly divided into two categories according to the biological mechanism: ectosomes (also known as microvesicles, microparticle) and exosomes (<200 nm in diameter).16 Ectosomes are formed by direct budding through the plasma membrane. The biogenesis process involves three coordinated mechanisms: (1) phospholipid bilayer reorganization driven by phosphatidylserine externalization, (2) calcium influx-triggered cytoskeletal disassembly through cofilin activation,28 and (3) arrestin domain-containing protein 1 (ARRDC1) mediated the recruitment of the ESCRT-I subunit tumor susceptibility gene 101 (TSG101), which promotes the assembly of vacuolar protein sorting-associated protein 4 (Vps4) ATPase to finalize vesicle scission.14 In contrast, exosomes originated from endosomes and multivesicular bodies (MVBs), formed early endosomes through endocytosis, and were released after fusion of MVBs with plasma membrane. Their cargo was loaded using mechanisms that were both dependent and independent of ESCRT.14

Furthermore, distinct categories of EVs emerge during particular cellular processes. For instance, apoptotic vesicles originate from apoptotic cells.29,30 Migrasome biogenesis is a mechanochemical process caused by tension from cell migration, stabilized by tetraspanin 4 (TSPAN4)-cholesterol complexes,31 and regulated by the PI (4,5) P2 /Rab35/integrin pathway32 (Figure 1). Some EVs also contain special cargo, such as mitochondria, called mitochondria-contained EVs (EV-Mito), which contain intact mitochondria or mitochondrial derived vesicles. EV-Mito is released through the exosomal pathway mediated by multivesicular bodies or the ectosomal pathway involving vesicle formation from the plasma membrane.33 Mechanisms of EVs biogenesis are described in detail in the articles by Andrew Dixson et al.13 It is worth noting that at present, there is a lack of specific molecular markers and standardized separation technology, so this paper uses EVs as a generalized term.

Figure 1 EVs classification and biogenesis. Ectosomes originate by directly budding outward and separating from the plasma membrane, while exosomes are released into the extracellular space through a three-step process: Initially, early endosomes are formed through the inward budding of the plasma membrane. These early endosomes then transform into late endosomes, eventually leading to the creation of MVBs. Subsequently, MVBs combine with the plasma membrane, causing the release of exosomes or they fuse with lysosomes, which leads to degradation. While apoptotic bodies are released by cells undergoing programmed cell death, migrasomes are vesicles that appear on the retraction fibers of migrating cells. These EVs are encased in a phospholipid bilayer and act as carriers for transporting a variety of cargoes such as lipids, proteins, nucleic acids, and metabolites. (Figure created with Figdraw).

Although we have a preliminary understanding of the biogenesis pathways of EVs, the specific biogenic processes of hucMSCs-EVs and their precise regulatory mechanisms for cargo sorting remain a “black box”.34 The lack of this knowledge directly leads to our inability to artificially and efficiently produce hucMSCs-EVs carrying specific therapeutic agent. The functional specificity of different subtypes of EVs is also difficult to define due to limitations in their isolation techniques, which poses fundamental challenges for standardized production and large-scale applications.35

Characteristics of MSC-EVs

MSCs can be obtained from different tissues, such as umbilical cord, bone marrow, and adipose tissue and so on. These MSC-EVs from different sources share both common and unique characteristics.

Shared Characteristics of hucMSCs-EVs with Other MSC-EVs

HucMSCs-EVs share basic molecular and functional similarities with EVs derived from other MSC sources, such as adipose tissue (ADSC-EVs), bone marrow (BMSC-EVs), and placenta (PMSC-EVs). All MSC-EVs contain essential EVs markers, including CD9, CD63, CD81, TSG101, and Alix,36,37 as well as MSC-specific surface markers like CD73, CD90, and CD105.38 These EVs usually carry bioactive molecules such as growth factors like VEGF and HGF, cytokines, and regulatory miRNAs, which contribute to their roles in anti-apoptosis, immunomodulation, and tissue repair.39–41

The analysis of Reactome pathways reveals common roles in essential biological processes, including platelet degranulation, extracellular matrix (ECM) organization, and insulin-like growth factor (IGF) transport, which support their regenerative abilities across various tissue types.40 For example, all MSC-EVs promote angiogenesis, reduce inflammatory responses, and improve cell survival through conserved signaling pathways such as PI3K-AKT and TGF-β.42,43

MSC-EVs from all sources also present organizational advantages, including stable phenotypes, lower immunogenicity than parental cells, and suitability for expandable production methods. These common characteristics highlight their shared potential as cell-free therapeutics, while tissue-specific molecular differences improve their applications for targeted diseases. While MSC-EVs from different sources have core therapeutic functions, hucMSCs-EVs exhibit great efficacy in specific situations because of their unique molecular signatures and tissue origin.

Different Characteristics of hucMSCs-EVs Compared to Other MSC-EVs

Despite shared core markers, MSC-EVs exhibit tissue-specific molecular features. Proteomic and transcriptomic analysis identified uniquely expressed proteins and miRNAs in MSC-EVs from different tissues. Among them, hucMSCs-EVs possess a greater number of identified protein markers (n=1393) compared to those from adipose tissue (n=21) and bone marrow (n=56). The protein markers of hucMSCs-EVs displayed in Figure 2B were further screened through bioinformatics analysis and are involved in key signaling pathways, highlighting a functionally distinct molecular signature. HucMSCs-EVs also demonstrate a unique miRNA expression profile (n=94), which is different from that of BMSC-EVs (n=134) and ADSC-EVs (n=689) (Figure 2A and B).40,44 These specific molecular features determine their applications. BMSC-EVs are mainly used in diseases such as osteoporosis and osteolysis. ADSC-EVs are mainly used for osteoarthritis and other joint diseases. HucMSCs-EVs are mainly used in female reproductive system diseases, diabetes (Figure 2B and C).40 The relevant content is described in detail in the articles by Zuo Ding et al and Sungho Shin et al40,44 Unfortunately, the source of these tissue-specific molecular features is not yet fully understood. Is it due to inherent differences in parental cells or the influence of extraction and culture conditions? This heterogeneity is not only the basis for its unique therapeutic potential, but also the main source of inter batch differences and unpredictable efficacy. At present, there is a lack of gold standard biomarkers that can clearly distinguish EVs from different tissue sources or functional subtypes, which seriously hinders the development of precision treatment strategies based on EVs.

Figure 2 Molecular characteristics and applications of MSC-EVs from different sources. (A) The Venn diagram of proteins in MSC-EVs derived from adipose (AD), bone marrow (BM), placenta (PL), and Wharton’s-jelly (WJ). Reproduced with permission from reference.44 Copyright 2021, MDPI; (B) Applications, protein and miRNA markers of each MSC-EVs. Reproduced with permission from reference.40 Copyright 2024, Elsevier; (C) The Sankey visualization of the effects of each MSC-EVs on different diseases and their target organs. Reproduced with permission from reference.40 Copyright 2024, Elsevier.

Applications of hucMSCs-EVs

MSCs-derived EVs show unique biological properties depending on their tissue origin. Although all MSC-EVs share common therapeutic functions, a growing body of evidence suggests that hucMSCs-EVs exhibit superior efficacy in tissue damage repair and certain oncology therapies. This prominence can be attributed to their parent cells, which have higher proliferation, lower immunogenicity, and better angiogenesis and immune regulatory abilities compared to other adult MSC sources.17,19,45 These inherent advantages make hucMSCs-EVs a particularly promising candidate for clinical translation. HucMSCs-EVs exert therapeutic potential through multiple common core mechanisms and unique mechanisms in different disease models.19,25,27,46–49 These core mechanisms include immunomodulation, autophagy regulation, regulation of cell survival, proliferation, and migration, metabolic regulation, and angiogenesis. This unified mechanism framework emphasizes the multifunctionality of hucMSCs-EVs as “biological nano-therapeutics” and provides information for the rational design of future EV-based solutions. These findings not only provide a theoretical foundation for understanding the regenerative medicine and anti-tumor value of hucMSCs-EVs, but also establish a mechanistic basis for their clinical translation. The following sections will present the therapeutic applications of hucMSCs-EVs across organ systems, dissecting their mechanisms in regenerative medicine and oncology (Figure 3).

Figure 3 HucMSCs-EVs exert therapeutic effects across various organ systems through diverse mechanisms. HucMSCs-EVs exhibit therapeutic effects on diseases related to eight systemic organ systems, including the nervous, locomotor, respiratory, digestive, urinary, reproductive, circulatory, and hormonal systems. These effects are mediated through multiple mechanisms that contain immunomodulation, antioxidant activity, autophagy regulation, mitochondrial protection, regulation of cell survival, proliferation, and migration, metabolic regulation, and angiogenesis. (Figure created with Figdraw).

Nervous SystemRegenerative EffectsTraumatic Brain Injury

Traumatic brain injury (TBI) remains a leading cause of global morbidity and mortality, with an estimated annual incidence of 27–69 million cases.50 It involves immediate mechanical damage to the brain, followed by secondary damage caused by inflammation, oxidative stress, and neuronal apoptosis, which worsens brain function.51 Despite advances in acute care, survivors often face cognitive impairment, motor dysfunction, and psychiatric disorders, imposing substantial socioeconomic burdens.51,52 Current therapies mainly focus on symptom alleviation and surgical interventions (eg, decompressive craniectomy), but these approaches fail to repair brain damage or promote functional recovery.52 Pharmaceuticals like anti-inflammatory drugs, neurotrophic factors such as NGF, and antioxidants, face challenges including poor blood-brain barrier (BBB) penetration, systemic toxicity, and short therapeutic windows. Moreover, advanced monitoring techniques (eg, microdialysis, cerebral oxygenation) are complicated and not yet standardized for personalized care.53–55 The emergence of hucMSCs-EVs has brought hope to TBI patients because of their small size, natural BBB permeability, and ability to modulate neuroinflammation, angiogenesis, and neuroprotection properties that make them ideal candidates for TBI repair.56,57

HucMSCs-EVs demonstrate strong neuroprotective and regenerative effects in TBI by combining anti-inflammatory, anti-apoptotic, pro-neurogenic, and angiogenic mechanisms. A key pathway suppresses neuroinflammation: hucMSCs-EVs inhibit NF-κB signaling, reduce the expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and shift microglial and astrocyte activation from a pro-inflammatory (M1/A1) to an anti-inflammatory (M2/A2) phenotype, thereby limiting secondary neuronal damage.18,46 Concurrently, hucMSCs-EVs prevent cell apoptosis and enhance neurogenesis, promoting neuronal survival.46 Moreover, hucMSCs-EVs have been found to promote angiogenesis.19 This capability facilitates blood supply restoration in the injured brain tissue, which is essential for supporting tissue repair and neurological recovery. The bioactive cargo within hucMSCs-EVs play a crucial role in these effects. For instance, miR-21 helps to suppress excessive activation of microglia, thus reducing neuroinflammation,58 while miR-146a-5p maintains neuronal integrity by specifically silencing pathways in neurotoxic astrocytes.59,60 Additionally, hucMSCs-EVs carry functional proteins like PINK1, which enhances mitophagy to alleviate oxidative stress and stabilize cellular homeostasis.61 Complementing these pathways, hucMSCs-EVs alleviate intracranial pressure and prevent further damage by reducing cerebral edema.61 Together, these effects create a healing microenvironment in the brain, leading to better recovery in TBI models. Unfortunately, the treatment of hucMSCs-EVs for TBI has only animal trials, not yet entered the human stage.

Stroke

Stroke remains a leading global cause of mortality and disability, with approximately 11.9 million new cases and 7.3 million deaths each year.62 According to the Global Burden of Disease Study, stroke imposes a significant socioeconomic burden because of its high incidence, recurrence rates, and long-term disabilities.63 Current therapeutic strategies like intravenous thrombolysis (eg, recombinant tissue plasminogen activator, rt-PA) and endovascular thrombectomy, are restricted by narrow treatment windows (≤4.5 hours for rt-PA) and low recanalization rates (30% for large vessel occlusions).64–66 Additionally, rt-PA has a significant risk of causing hemorrhagic transformation, occurring in 8.94–46% of cases, which limits its use in clinical.67–69 These therapies also fail to manage post-ischemic secondary neurodegeneration, BBB disruption, or neuroinflammation, resulting in survivors having ongoing neurological deficits.70 To bridge this gap, researchers are exploring hucMSCs-EVs, which show promise in repairing stroke induced damage through different mechanisms.

HucMSCs-EVs exert neuroprotective and regenerative effects in stroke through various molecular mechanisms. For example, they reduce neuroinflammation by delivering miR-146a-5p, which inhibits the IRAK1/TRAF6/NF-κB inflammatory pathway. This reduces pro-inflammatory cytokines like IL-6 and TNF-α and shifts microglia to the protective M2 state, which helps to prevent more neuronal damage.47 HucMSCs-EVs also inhibit ferroptosis by transferring miR-214-3p, which targets key regulators like GPX4 and ACSL2. This preserves mitochondrial integrity, reduces oxidative stress in neurons, and restores BBB integrity in hemorrhagic stroke models.71 Besides, through miR-664a-5p, hucMSCs-EVs regulate Adaptor-Associated Kinase 1 (AAK1), a kinase involved in clathrin-mediated endocytosis and NF-κB signaling pathway. By suppressing AAK1, they inhibit NF-κB-driven inflammatory cascades, leading to smaller infarct volumes and better functional recovery in ischemic stroke models.72 Animal experiments demonstrate that hucMSCs-EVs injections via veins or the nose reduces brain damage and enhances neurological function. According to a meta-analysis of 38 randomized controlled animal experiments, hucMSCs-EVs markedly enhance both movement and cognitive scores.73 In a rat model of focal cerebral ischemia, MSC-EVs enhance neurogenesis and angiogenesis along the edge of the infarct. They can also improve synaptic transmission, long-term potentiation, and cognitive impairment after transient cerebral ischemia in mice. In a mouse model of focal cerebral ischemia, the administration of BMSC-EVs induced long-term neuroprotection, enhanced angiogenesis and neurogenesis, and facilitated better recovery of motor coordination. HucMSCs-EVs may have a similar effect as they can also promote angiogenesis and neurogenesis, thereby repairing damaged brain tissue.19,46,74 The diverse therapeutic capabilities of hucMSCs-EVs enable the possibility of functional recovery after stroke. Thus, hucMSCs-EVs have emerged as a groundbreaking therapeutic candidate. To date, research on hucMSCs-EVs for stroke has not progressed beyond the preclinical phase, with efficacy data derived exclusively from animal experiments.

Alzheimer’s Disease

Alzheimer’s disease (AD), affecting over 55 million people globally, is the most prevalent neurodegenerative disorder, characterized by progressive cognitive decline associated with β-amyloid (Aβ) plaque accumulation, neurofibrillary tau tangles, and chronic neuroinflammation.75 The therapies approved by the FDA, such as acetylcholinesterase inhibitors like donepezil and NMDA receptor antagonists like memantine, only relieve symptoms without altering disease progression.76,77 Although new anti-Aβ monoclonal antibodies such as aducanumab show plaque removal, their clinical advantages are debated due to limited BBB penetration and inability to tackle issues like synaptic loss and neuroinflammation.78–80 The absence of effective treatments has spurred research into novel strategies aimed at the complex pathophysiology of AD.

HucMSCs-EVs emerge as promising candidates, utilizing their innate ability to cross the BBB, immunomodulatory and neuroprotective properties. They demonstrate multifaceted therapeutic mechanisms against AD, targeting its core pathological hallmarks, including Aβ plaque accumulation, neuroinflammation, mitochondrial dysfunction, and neuronal apoptosis. The regulation of Aβ metabolism is a key mechanism, with hucMSCs-EVs enhancing the production of Aβ-degrading enzymes like neprilysin (NEP) and insulin-degrading enzyme (IDE) to promote plaque clearance.48,81 They also modulate secretase activity by upregulating α-secretase and downregulating β-secretase (BACE1), thereby reducing Aβ production.48,82 These effects are further amplified in engineered EVs, such as those transfected with miR-29c mimics or loaded with NEP, which show enhanced Aβ degradation in animal models.48 By reprogramming microglial activation, hucMSCs-EVs also demonstrate significant immunomodulatory effects. They reduce brain inflammation by changing microglia from M1 phenotype to M2 phenotype, suppressing pro-inflammatory cytokines like TNF-α and IL-1β, while upregulating anti-inflammatory mediators such as TGF-β and IL-10.48,81 This immunomodulation is mediated by EVs with miR-146a and TGF-β1, which attenuate neuroinflammatory cascades and protect neurons from damage.48 Mitochondrial dysfunction, a critical contributor to AD progression, is addressed through EV-mediated mitochondrial transfer. HucMSCs-EVs deliver functional mitochondria to neurons, restoring membrane potential, enhancing ATP production, and reducing oxidative stress, thereby preventing apoptosis and improving neuronal survival.83 In addition, suppressing miR-211-5p in hucMSCs-EVs increases NEP expression, which facilitates Aβ clearance and protects neurons.84 Other miRNAs target pathways linked to synaptic plasticity, apoptosis, and inflammation, further amplifying therapeutic efficacy.48,82 Studies show that delivering hucMSCs-EVs through the nose helps them reach brain areas like the hippocampus, improving memory in animals.85 By acting as a “multi-action therapy”, hucMSCs-EVs target Aβ, tau proteins, inflammation, mitochondrial dysfunction, and synaptic loss, which makes them superior to traditional single-target treatments. It is encouraging that hucMSCs-EVs have not only undergone animal trials for the treatment of AD, but have also entered human clinical trials and are in Phase I (Table 1).

Table 1 Registered Clinical Trials with hucMSCs-EVs Interventions on Clinicaltrials.gov (http://www.clinicaltrials.gov/)

Parkinson’s Disease

Parkinson’s disease (PD), the second most prevalent neurodegenerative disorder globally, affects over 11.77 million people and is characterized by progressive dopaminergic neuron loss in the substantia nigra and pathological α-synuclein aggregation.86,87 The clinical hallmarks—tremor, bradykinesia, rigidity, and postural instability—are compounded by non-motor symptoms such as cognitive decline and autonomic dysfunction, which collectively reduce quality of life.88 Pharmacological interventions, mainly focus on dopamine replacement (eg, levodopa), which can provide transient relief, but are plagued by motor fluctuations (eg, dyskinesias and “on-off” phenomena) and non-motor side effects, such as orthostatic hypotension and neuropsychiatric complications.89–91 Long-term use of dopamine agonists, although reducing reliance on levodopa, can bring risks such as impulse control disorders and cardiac valvulopathy.92,93 Surgery (eg, deep brain stimulation) offer sustained symptom control but is invasive, costly, and unsuitable for advanced patients with cognitive impairments.94 No treatments yet slow or reverse PD progression, highlighting the need for new strategies.

HucMSCs-EVs fight PD by targeting dopaminergic neuron loss, neuroinflammation, and α-synuclein aggregation. They cross the BBB and accumulate in damaged regions, such as the substantia nigra and olfactory bulb, where they are internalized by neurons, microglia, and astrocytes to exert localized repair.95–97 Neuroprotection is achieved through multiple pathways: hucMSCs-EVs enhance the viability of dopaminergic neurons by increasing tyrosine hydroxylase-positive cells in the substantia nigra pars compacta, and restore olfactory function by improving neuronal activity in the olfactory bulb.95,96 They also induce autophagy, upregulating LC3B-II/I and Beclin-1 while downregulating p62, thereby clearing toxic protein aggregates and alleviating 6-hydroxydopamine (6-OHDA)-induced apoptosis.98

The anti-inflammatory properties of hucMSCs-EVs are critical in modulating PD-associated neuroinflammation. By attenuating microglial and astrocytic activation, these EVs reduce pro-inflammatory cytokines (eg, TNF-α, IL-1β) and inhibit the PI3K/Akt-mediated NF-κB/NLRP3 pathway, thereby suppressing pyroptosis and creating a neuroprotective microenvironment.96,97 Additionally, engineered hucMSCs-EVs hybridized with antioxidants like baicalein or oleuropein disrupt α-synuclein fibrillation, reduce reactive oxygen species (ROS) and nitric oxide (NO) levels, and protect BBB integrity, fighting against oxidative stress and neurotoxicity.99,100

HucMSCs-EVs further enhance neuronal survival by modulating multiple signaling pathways. SATB1 upregulation triggers the Wnt/β-catenin pathway, which promote neurogenesis and neurite outgrowth while inhibiting excessive autophagy.96 Engineered EVs loaded with brain-derived neurotrophic factor (BDNF) maintain neuronal cytoskeletons by regulating microtubule-associated protein 2 (MAP2) and phosphorylated tau, and activate the Nrf2 antioxidant pathway to combat ferroptosis and oxidative damage.100,101 Through the combination of autophagy induction, α-synuclein clearance, oxidative stress reduction, and pathway modulation, hucMSCs-EVs present a powerful multi-target approach to combat PD. Current therapeutic applications of hucMSCs-EVs for PD remain confined to animal models, with no clinical trials reported to date.

Multiple Sclerosis

Multiple sclerosis (MS), a chronic autoimmune disorder affecting 2.8 million people globally, is characterized by demyelination, neuroinflammation, and axonal damage in the central nervous system (CNS), leading to progressive neurological disability.102 Current disease-modifying therapies (DMTs) such as β-interferons, dimethyl fumarate, and anti-CD20 monoclonal antibodies, mainly target peripheral immune activation to reduce recurrence rates, but have limited efficacy in progressive MS and fail to promote CNS repair or remyelination.103 Furthermore, long-term immunosuppression increases infection risks, while high-dose corticosteroids used for acute recurrence can cause metabolic and osteoporotic complications.104–106 These unsatisfied needs fully demonstrate that there is an urgent need to develop therapies that can combine immunomodulation with neuroprotection and regeneration.

In MS, hucMSCs-EVs demonstrate therapeutic potential by targeting autoimmune dysregulation, neuroinflammation, and demyelination. The core of its efficacy lies in immunomodulation: hucMSCs-EVs suppress pathogenic T-cell proliferation, particularly CD4+CD25− conventional T cells, while enhancing regulatory T cell (Treg) expansion, including CD4+CD25+Foxp3+ populations, to restore immune tolerance.107,108 This is achieved through dual cytokine regulation, which downregulate pro-inflammatory mediators (IFN-γ, IL-17, TNF-α, IL-1β, IL-6) and upregulate anti-inflammatory cytokines (IL-10, TGF-β, IL-4). This rebalances the Th17/Treg axis and reduces neuroinflammation in both in vitro MS models and experimental autoimmune encephalomyelitis (EAE) mice.107–109

It is crucial that hucMSCs-EVs promote remyelination by delivering miR-23a-3p, which activates the PI3K/Akt pathway and suppresses Tbr1/Wnt pathway in oligodendrocyte precursor cells (OPCs), which promotes their differentiation into mature myelinating oligodendrocytes.110 Meanwhile, these EVs enhance myelin basic protein (MBP) expression, reduce demyelinated lesions in spinal cord tissues, and improve neurological function in EAE models.15,109 By modulating microglial polarization to anti-inflammatory M2 phenotype, this approach enhances their regenerative potential, helping to resolve chronic inflammation and promote tissue repair.15

In addition, hucMSCs-EVs enhance the inhibitory function of Treg by upregulation lymphocyte-activation gene 3 (Lag-3) in Foxp3+CD4+ T cells, thereby inhibiting immune cell proliferation and cytokine storm in EAE.111 Unlike traditional DMTs that lack remyelination or neuroprotective effects, hucMSCs-EVs are a “multi-drug platform” that can simultaneously inhibit autoimmune attacks, reduce neuroinflammation, and stimulate CNS repair. Preclinical studies have emphasized their superior efficacy compared to whole MSCs, with peptide modified EVs targeting the CNS showing enhanced biodistribution and BBB penetration.15 Despite promising results in animal studies, hucMSCs-EVs-based interventions for MS have not yet advanced to human clinical trials.

Spinal Cord Injury

Spinal cord injury (SCI), a severe condition impacting over 27 million people worldwide, causes permanent loss of movement, sensation, and autonomic dysfunction. This occurs not only from primary mechanical damage but also from secondary pathological cascades involving inflammation, glial scar formation, and axonal degeneration.112–114 Beyond physical disability, SCI patients face heavy mental stress, with suicide risks 2–37 times higher than healthy individuals.115 Current clinical interventions, such as surgical decompression, methylprednisolone pulse therapy, and rehabilitation mainly manage early-stage problems but cannot address neural regeneration or functional recovery.116 Although steroids like methylprednisolone reduce swelling, long-term use can cause serious side effects (eg, immunosuppression, osteoporosis) without healing nerves.117 New approaches using stem cells show limited promise in animal studies but struggle with poor survival, uncontrolled differentiation, and tumorigenic risks.118 These challenges bring attention to the urgent need for innovative strategies to repair damaged nerve environments.

HucMSCs-EVs exhibit diverse therapeutic potential in SCI repair by simultaneously addressing inflammation, angiogenesis, neurogenesis, apoptosis, autophagy, and microenvironment modulation.

Immunomodulatory

HucMSCs-EVs alleviate neuroinflammation by switching macrophage/microglia from the M1 to M2 phenotype. This shift decreases pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ) and increases anti-inflammatory mediators (IL-4, IL-10).119–124 The conversion of AMP to adenosine is limited by CD73 (ecto-5′-nucleotidase), which is the rate-limiting ecto-enzyme in extracellular AMP hydrolysis. CD73+ hucMSCs-EVs, promote adenosine production via ATP hydrolysis, which activates the A2bR/cAMP/PKA pathway to enhance M2 polarization.120 The 4D-culture, which characterized by extended 3D-culture time and enhanced cell-microenvironment interaction that forms an MSC-favorable niche generates EVs that upregulate IGFBP2/EGFR, activate STAT3, and suppress neuroinflammation.121 IL-4-engineered EVs inhibit PDCD4 by delivering miR-21-5p, further promoting M2 polarization.122 Quercetin-loaded EVs suppress TLR4/NF-κB signaling, reducing cytokine release.123 A supportive regenerative microenvironment is created through immunomodulatory.

Angiogenesis

HucMSCs-EVs enhance vascular repair by stimulating endothelial cell migration and tube formation. By transferring miR-27a-3p, CD146+CD271+ EVs decrease DLL4 expression, which promote angiogenesis and functional recovery in vivo.125 Hypoxia-preconditioned EVs further amplify angiogenic signaling in MSCs, restoring blood supply to injured tissue.126 The increase in CD31+ endothelial cell proliferation and vascular density at lesion sites confirm their pro-angiogenic efficacy.125,126

Neurogenesis and Axonal Regeneration

HucMSCs-EVs enhance the expression of neuronal markers such as NF200, MBP, GAP43, synaptophysin and PSD95, which promote remyelination, synaptogenesis, and axonal growth.124,127–130 miR-126-modified EVs enhance neurogenesis by increasing neural progenitor cells and neurons,131 while miR-29b-3p targets PTEN to activate the Akt/mTOR pathway, promoting nerve repair.132 EVs also activate endogenous neural stem cells (NSCs) via ERK1/2 signaling, driving their proliferation and differentiation.128 Scaffold-coupled EVs, such as collagen-paclitaxel hybrids, synergistically recruit NSCs and enhance neural network reconstruction.133

Anti-Apoptotic and Pro-Autophagic Effects

HucMSCs-EVs inhibit apoptosis by downregulating Bax, cleaved caspase-3, and p75NTR while upregulating Bcl-2.124,126–129,131,134 Under the influence of micro electrical fields, hucMSCs-EVs transport lncRNA-MALAT1 to target miR-22-3p, elevating SIRT1 and AMPK phosphorylation to inhibit apoptosis.134 In addition, EVs maintain cell homeostasis and survival by enhancing autophagy.134

Microenvironment Regulation and Barrier Repair

HucMSCs-EVs restore blood-spinal cord barrier (BSCB) integrity by downregulating endothelin-1 (ET-1) and upregulating junction proteins (ZO-1, β-catenin, occludin, claudin-5).135,136 RGD−CD146+CD271+ hucMSCs-EVs modulate endothelial cells by suppressing the miR-501-5p/MLCK axis, stabilizing tight junctions and reducing vascular leakage.136 HucMSCs-EVs also attenuate oxidative stress and glial scarring through miR-138-mediated NLRP3-caspase1 and Nrf2-keap1 pathway.137 Quercetin-loaded EVs limit scar formation by inhibiting JAK2/STAT3 and A1 astrocyte activation.123

Targeted Delivery and Clinical Translation

After intranasal administration, functionalized EVs like RGD-modified EVs specifically accumulate, improving therapeutic accuracy.136 Tannic acid hydrogels enable sustained EVs release, reducing ROS and inflammation while preserving motor and urinary function.138 Early-phase trials demonstrate intrathecal hucMSCs-EVs safety and functional improvement in subacute SCI, highlighting translational potential.129 Encouragingly, hucMSCs-EVs therapy for SCI has advanced beyond animal studies to initiate clinical trials in humans (Table 2).

Table 2 Registered Clinical Trials with hucMSCs-EVs Interventions on Irct.behdasht.gov.ir (https://irct.behdasht.gov.ir/)

Peripheral Nerve Injury

Peripheral nerve injury (PNI), commonly caused by physical trauma from accidents, natural disasters, wars, or surgical complications, affecting over one million individuals worldwide each year.139 Damage often results in permanent motor and sensory deficits due to slow axonal regeneration rates, Wallerian degeneration, and inadequate Schwann cell (SC) remyelination, particularly in large injury gaps (1 cm in rodents and 3 cm in humans).140,141 Current treatments include microsurgical suturing, autologous nerve grafting, and nerve conduits. While autografts remain the “gold standard” for bridging nerve gaps, they face limitations such as donor site damage, limited availability, and poor efficacy for long-gap injuries (>15 mm).139 Synthetic nerve conduits, such as BDNF-loaded chitosan-based biomimetic polymers, have shown promise in preclinical models by enhancing axonal regeneration and myelination. However, their efficacy as independent therapies is still limited by insufficient bioactivity and incomplete microenvironmental modulation.139 Complementary approaches, including electrical stimulation and traditional Chinese medicine techniques (eg, acupuncture, tuina), aim to accelerate nerve repair by promoting neurotrophic factor expression and reducing inflammation.142–144 However, these methods lack standardized protocols and exhibit variable outcomes depending on injury type and severity. These challenges highlight the urgent need for therapies that simultaneously enhance axonal regeneration, modulate the inhibitory microenvironment, and prevent muscle atrophy during prolonged recovery.

For PNI, hucMSCs-EVs show multifaceted regenerative potential, resolving critical challenges such as slow axonal regeneration, SC dysfunction, neuroinflammation, and muscle atrophy. HucMSCs-EVs promote SC proliferation, migration, and differentiation by upregulating migration-related genes (MMP9, MMP13), adhesion molecules (N-cadherin, Integrin β1), and cell cycle regulators (PCNA, Cyclin E1).145–147 This process is achieved through transferring miR-21, which activates cell growth pathways (PI3K/Akt/mTOR) while blocking stress signal (MAPK), enhancing SC-mediated axonal elongation and remyelination.145,146 HucMSCs-EVs also stimulate SC to secrete neurotrophic factors like NT-3 and BDNF, creating a regenerative microenvironment.147,148 The repair process is further supported by immunomodulation, as hucMSCs-EVs reduce pro-inflammatory cytokines (IL-6, IL-1β) while elevate anti-inflammatory IL-10, resolving chronic inflammation that impedes regeneration.149,150 They also enhance angiogenesis, as evidenced by increased CD31+ endothelial cells in regenerated nerves, improving oxygen and nutrient delivery to injury sites.150 To combat muscle atrophy, hucMSCs-EVs suppress muscle-specific ubiquitin ligases (Fbxo32, Trim63) by delivering miR-23b-3p, maintaining muscle mass and function during long-term recovery.151 Through hypoxic pretreatment, hucMSCs-EVs are enriched with factors that promote regeneration, enhance SC migration, and neurotrophic factor secretion.147 Engineered delivery systems, such as dual-responsive hydrogels or 3D-printed nerve conduits loaded with decellularized ECM (dECM)-encapsulated EVs, enable sustained release and spatial targeting, improving functional recovery in rodent sciatic nerve models.145,150,152 Combinatorial methods, such as combining hucMSCs-EVs with olfactory ensheathing cells (OECs), can synergistically promote BDNF secretion and SC survival in hypoxic conditions, accelerating axonal reconnection.148 These studies highlight the potential of hucMSCs-EVs as a cell-free, multi-targeted therapy for PNI. HucMSCs-EVs improve neurological diseases through multiple mechanisms such as immune regulation, angiogenesis, and nerve regeneration. HucMSCs-EVs treatment for PNI is currently supported only by animal-level evidence, human trials are lacking. The specific molecular pathways are summarized in Figure 4 and Table 3.

Figure 4 The effects of hucMSCs-EVs carrying different cargoes on different nervous system diseases. The red colored upward arrows indicate promotion or upregulation, while the green colored downward arrows indicate inhibition or downregulation.

Table 3 Molecular Mechanism of hucMSCs-EVs in Treating Different Diseases in the Nervous System

Anti-Tumor EffectsGlioblastoma

Glioblastoma (GBM) is the most invasive primary brain tumor, and despite standard treatments such as surgery, radiation therapy, and chemotherapy, the prognosis is still poor. The BBB restricts drug delivery, tumor heterogeneity promotes therapy resistance, and the immunosuppressive microenvironment further limits treatment efficacy.153–156

HucMSCs-EVs have emerged as promising nanocarriers for GBM therapy due to their tumor accumulation, biocompatibility, and ability to cross the BBB. These vesicles mediate therapeutic effects through multiple mechanisms, including direct induction of apoptosis, cell cycle arrest, immunomodulation, and targeted delivery of therapeutic nucleic acids or drugs. Yueh et al demonstrated that miR-124-loaded hucMSCs-EVs downregulate CDK4 and CDK6, leading to G1 phase arrest and apoptosis in GBM cells. Additionally, these EVs modulate the tumor microenvironment by enhancing T-cell activation and dendritic cell maturation, while suppressing regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), thereby promoting an anti-tumor immune response.153 Moreover, hucMSCs-EVs serve as efficient vehicles for enzyme/prodrug systems. Tibensky et al showed that EVs derived from hucMSCs expressing the yeast cytosine deaminase::uracil phosphoribosyl transferase (yCD::UPRT) gene can convert the prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) within tumor cells, leading to significant tumor growth inhibition and even complete regression in rodent GBM models.155 Del Fattore et al reported that hucMSCs-EVs inhibit GBM cell proliferation and induce apoptosis. When hucMSCs-EV are loaded with chemotherapy drugs such as vincristine, this anti-tumor effect is further enhanced and drug delivery and cytotoxicity are improved.156 However, the dual role of MSC-EVs in GBM must be carefully considered. Pavon et al highlighted that although hucMSCs exhibit strong bias toward CD133+ GBM stem cells through chemokine signaling pathways such as MCP-1/CCL2 and SDF-1/CXCL12, their subsequent recruitment may inadvertently support tumor proliferation and invasion through EVs mediated intercellular communication.154 In summary, hucMSCs-EVs represent a multifunctional platform for GBM therapy through targeted RNA interference, enzyme/prodrug activation, and immunomodulation. The specific molecular pathways are summarized in Figure 4 and Table 3.

Locomotor SystemRegenerative EffectsBone-Related Disorders

Bone-related disorders, including osteoporosis, fracture nonunion, and osteonecrosis, affect over 200 million people worldwide and impose a significant global health burden.157 These conditions are often exacerbated by dysregulated bone metabolism, where imbalances in osteoblast-osteoclast activity and impaired energy metabolism pathways disrupt bone remodeling, leading to structural fragility and delayed healing.158,159 Current therapeutic strategies, such as bisphosphonates, parathyroid hormone analogs, and surgical interventions, remain limited.160 Drugs can cause systemic side effects and fail to address the underlying metabolic dysfunction,161 while surgeries like bone grafts may struggle with poor tissue integration or infections.162,163 Using a patient’s own stem cells for treatment shows potential but faces hurdles like poor cell survival, inconsistent differentiation, and ethical concerns.164 This highlights the urgent need for therapies that enhance osteogenesis, suppress pathological osteoclast activity, and modulate the inflammatory microenvironment.

HucMSCs-EVs for Osteoporosis

In osteoporosis, hucMSCs-EVs inhibit BMSC apoptosis via the miR-1263/Mob1/Hippo signaling pathway, restore osteoblast-adipocyte differentiation balance, and maintain bone homeostasis.165,166 These EVs also carry proteins like CLEC11A, which is a pro-osteogenic protein that push BMSC to become osteoblasts instead of adipocytes, while suppressing the formation of osteoclasts, effectively reducing bone resorption and bone marrow fat accumulation.167 Although some potencies have been achieved in animal studies, hucMSCs-EVs-based interventions for osteoporosis have not yet entered to human clinical trials.

HucMSCs-EVs for Fracture

For bone regeneration and fracture healing, hucMSCs-EVs enhance osteogenesis by upregulating osteogenic proteins such as RUNX2, ALP, BMP-2, and OCN, which promote the deposition of mineralized matrix and the formation of calcified nodules in BMSC and osteoblast progenitor.45,168,169 They also promote angiogenesis by activating HIF-1α and VEGF in endothelial cells, stimulating proliferation, migration, and tube formation, which improves oxygen and nutrient supply to injured areas.169–171 Notably, hucMSCs-EVs derived miR-23a-3p targets PTEN to activate the AKT pathway and linking osteogenesis with osteogenesis and angiogenesis for vascularized bone repair.169 By integrating hucMSCs-EVs with biomaterials, including hyaluronic acid hydrogels and nanohydroxyapatite scaffolds, the delivery efficiency of osteoinductive factors like rhBMP-2 is enhanced, accelerating regeneration in osteoporotic defects.172–174 So far, researches on hucMSCs-EVs therapy for fracture have remained limited to animal models and lack human trials.

HucMSCs-EVs for Cartilage and Intervertebral Disc Repair

In cartilage and intervertebral disc repair, hucMSCs-EVs modulate immune microenvironments by reducing inflammation and suppressing ECM degradation. They enhance mitochondrial function in chondrocytes via transferring glycolytic enzyme and alleviate endoplasmic reticulum stress in nucleus pulposus cells by activating AKT/ERK pathway, thus reducing apoptosis and fibrosis.171 HucMSCs-EVs treatment for cartilage and intervertebral disc repair is currently supported only by animal-level evidence, human trials are lacking.

HucMSCs-EVs for Periapical Periodontitis

The anti-inflammatory properties further contribute to bone repair in inflammatory environments, such as periapical periodontitis, where hucMSCs-EVs reduce osteoclast activity and inflammatory cell infiltration, promoting alveolar bone regeneration.45 Notably, therapeutic development of hucMSCs-EVs for periapical periodontitis has transitioned from animal validation to active Phase II human clinical evaluation (Table 1).

These studies indicate that hucMSCs-EVs are effective multi-target treatment for osteoporosis, fracture non-unions, cartilage and intervertebral discs, but strict trials are needed to verify long-term efficacy and safety.

Joint-Related Disorders

Joint-related disorders like osteoarthritis (OA), rheumatoid arthritis (RA) impact billions worldwide,175,176 leading to chronic pain, disability, and irreversible cartilage loss caused by imbalanced inflammatory cascades, chondrocyte apoptosis, and ECM degradation.177,178 Existing treatments for joint diseases remain inadequate. Pharmacological interventions, including nonsteroidal anti-inflammatory drugs (NSAIDs), can alleviate symptoms but cannot prevent disease progression. Long term use carries risks of gastrointestinal, cardiovascular, and renal toxicity.179 Intra-articular injections of corticosteroids or hyaluronic acid offer transient benefits but require frequent administration due to rapid clearance, reducing patient compliance.180 Although total knee arthroplasty and other surgeries are effective for end-stage OA, they are invasive, costly, and associated with complications such as infection and incomplete functional recovery.181 These shortcomings emphasize the urgent need for therapies that simultaneously resolve inflammation, restore cartilage integrity, and modulate the synovial microenvironment.

HucMSCs-EVs for OA

HucMSCs-EVs show multiple healing abilities for OA by addressing inflammation, angiogenesis, cartilage regeneration, synovial microenvironment modulation.

Immunomodulation and Inflammation Resolution

HucMSCs-EVs reprogram immune cells to relieve synovial inflammation. They polarize macrophages from M1 to M2 phenotypes by suppressing NLRP3 inflammasome activation via miR-223 binding to NLRP3 mRNA20 and reducing METTL3-mediated m6A modification of NLRP3.21 This shift decreases synovial TNF-α, IL-1β, and IL-6 while elevating IL-10 and TGF-β, creating a regenerative microenvironment.