Neural Stem Cell-Derived Extracellular Vesicles Alleviate Inflammatory Responses in a Mouse Model of Atopic Dermatitis

Rheumatology and Immunology Immunology Bioengineering Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine
neural stem cells extracellular vesicles atopic dermatitis inflammation skin barrier immune regulation mouse model

I. Academic Background and Research Motivation

Atopic Dermatitis (AD) is a common chronic inflammatory skin disease characterized by eczematous lesions, intense itching, and impairment of skin barrier function. Its pathogenesis is highly complex, involving genetic susceptibility, epidermal barrier dysfunction, immune dysregulation, and environmental factors. Previous studies have shown that atopic dermatitis not only affects skin health but also significantly impairs patients’ quality of life, and is closely associated with comorbidities such as respiratory allergies, hypersensitivity reactions, and certain autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease).

Currently, the main treatment approaches for atopic dermatitis include corticosteroids and immunosuppressants, but these therapies generally provide only temporary relief and carry numerous side effects with long-term use (such as skin atrophy, pigment changes, systemic adverse reactions, etc.), making long-term clinical management of patients challenging. Therefore, there is a pressing need in the field to identify new mechanisms and intervention strategies.

Stem cells and their derivatives have attracted growing attention in recent years for their immunomodulatory and regenerative repair potential, especially mesenchymal stem cells (MSC), which have shown roles in immune regulation and skin barrier repair in animal experiments and some clinical studies. Extracellular vesicles (EVs), as nanoscale particles secreted by stem cells, carry various active factors such as proteins, mRNA, miRNA, and lipids, and are considered “messengers” in intercellular communication and regulation of inflammation. Some studies have confirmed that MSC-derived EVs can alleviate skin inflammation and promote epidermal repair in AD mouse models. However, the mechanism and potential of extracellular vesicles derived from neural stem cells (NSCs) in atopic dermatitis have not yet been systematically studied.

Therefore, this study addresses the current challenges in atopic dermatitis treatment and the need for novel therapeutic strategies by systematically investigating, for the first time, the regulatory roles and molecular mechanisms of neural stem cells and their derived extracellular vesicles in modulating AD-related inflammatory responses and skin repair, and further elucidates their molecular basis through proteomic analysis.

II. Source of the Paper and Author Information

The article, titled “neural stem cell-derived extracellular vesicles alleviate inflammatory responses in a mouse model of atopic dermatitis,” was completed by Seulbee Lee, Donghun Hyun, Yong Namkung, Boram Park, Byounggwan Lee, Junhyung Myung, and Sunghoi Hong, with Sunghoi Hong as the corresponding author. The authors are all affiliated with Korea University, a prestigious institution in the field of biomedical research in South Korea. The article was published in 2025 in an academic journal hosted by Oxford University Press (see original DOI: 10.1093/stmcls/sxaf034) and is open access, which greatly facilitates academic exchange.

III. Research Design and Technical Workflow Details

1. Overall Research Design and Main Workflow

This study is an original experimental investigation, and the workflow includes: - Preparation and isolation of neural stem cells, their conditioned media (CM), and extracellular vesicles (EVs) - Assessment of anti-inflammatory effects in vitro using inflammation models (human keratinocyte HaCaT cells, murine macrophage RAW264.7 cells) - Evaluation of clinical efficacy and pathological improvement in an in vivo mouse model of atopic dermatitis - Proteomic identification of NSC-EVs to explore underlying molecular mechanisms

1.1 Preparation and Isolation of Stem Cells and Extracellular Vesicles

The authors chose immortalized human neural stem cells (NSCs) as the stem cell source. Cell culture was performed using high-glucose Dulbecco’s Modified Eagle Medium (DMEM) + 10% fetal bovine serum or exosome-depleted serum under standard conditions. Conditioned media were collected when cells reached 90% confluency, centrifuged to remove cells and large debris, filtered through a 0.22 μm membrane, concentrated with a 100 kDa molecular weight cutoff centrifugal filter, and finally purified using an Exo-i proprietary column (an advanced nanovesicle purification technique that significantly enhances exosome purity). EV concentration was determined using a BCA protein assay to ensure experimental consistency.

1.2 Characterization of Extracellular Vesicles

The authors characterized the morphology and surface markers of the isolated extracellular vesicles. Morphological features were observed using field emission transmission electron microscopy (FE-TEM), while particle size distribution was measured with dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). On the protein level, Western blotting was used to detect classical EV markers CD63 and CD9, ensuring nanovesicle purity in line with international MISEV2018 standards. For cellular uptake experiments, PKH67 fluorescent labeling was employed, with confocal microscopy used to visualize real-time localization of particles after uptake by keratinocytes.

1.3 In Vitro Inflammation Model Experiments

  • HaCaT Keratinocyte Inflammation Model: Inflammation was induced with TNF-α and IFN-γ. HaCaT cells were treated with varying concentrations of NSC-CM (10%, 50%, 100%) and different doses of NSC-EVs (50μg, 100μg, 500μg). RT-PCR was used to detect mRNA expression of inflammatory cytokines (e.g., IL-6, TNF-α) and chemokines (e.g., RANTES, MCP-1, TARC); Western blot was used to examine NF-κB (total and phosphorylated) and IL-1β expression.
  • RAW264.7 Macrophage Inflammation Model: Inflammation was induced with LPS, with concurrent NSC-CM treatment. Analyses were similar to those above, including expression of inflammatory cytokines, chemokines, inflammatory enzymes (iNOS, COX-2), and NO release (measured by Griess reagent colorimetry).

1.4 In Vivo Atopic Dermatitis Mouse Model

  • The experimental animals were 8-week-old male NC/Nga mice, randomly divided into five groups: negative control, AD model, NSC-CM treatment group, NSC-EV treatment group, and tacrolimus positive control group. AD-like lesions were induced on the dorsal skin using 2,4-dinitrochlorobenzene (DNCB).
  • Each group was treated topically with PBS, NSC-CM, NSC-EVs, or tacrolimus daily for three weeks. Efficacy was evaluated through clinical scoring, photography, histopathological examination with H&E and toluidine blue staining (including quantification of epidermal thickness and mast cell infiltration).
  • A PKH67-labeled NSC-EV skin absorption experiment was conducted to demonstrate the nanovesicles’ ability to penetrate the skin barrier.

1.5 Proteomics Analysis

  • NSC-EVs were lysed, digested by FASP, labeled with TMT, fractionated by reverse-phase liquid chromatography, desalinated with C18 columns, and finally subjected to highly sensitive LC-MS/MS for protein identification.
  • Data analysis was performed using Proteome Discoverer software, with all results controlled at an FDR of less than 1% and only proteins with at least two unique peptides included. Analysis focused on the top 100 most abundant proteins, utilizing STRING (protein interaction database) and the R package circlize for protein interaction and functional enrichment analysis. Gene Ontology (GO) enrichment analysis was completed using ShinyGO, focusing on biological processes and cellular components.

2. Main Experimental Results and Data Details

2.1 Immunological Comparison Between NSC-CM and ADSC-CM

Antibody array analysis showed that, compared to adipose-derived mesenchymal stem cell conditioned media (ADSC-CM), NSC-CM contained significantly lower levels of inflammatory cytokines (such as IL-6, RANTES, the MCP family, etc.), indicating a higher degree of immunological “tolerance.”

2.2 Benefits in In Vitro Inflammation Models

  • HaCaT Experiment: 100% NSC-CM significantly downregulated the expression of key inflammatory factors such as IL-6, TNF-α, TARC, RANTES, and MCP-1. Immunoblotting revealed that NSC-CM inhibited NF-κB phosphorylation and IL-1β protein expression in a dose-dependent manner without affecting total NF-κB levels, suggesting negative regulation of inflammation via the NF-κB pathway.
  • RAW264.7 Experiment: NSC-CM effectively suppressed LPS-induced macrophage activation, as reflected by restoration of resting cell morphology, significant downregulation of inflammatory factors and enzymes, and reduced NO release. The inhibitory effect was also dose-dependent, with 100% NSC-CM being most effective.

2.3 Purity and Functional Identification of NSC-EVs

EVs were characterized by electron microscopy, DLS, and NTA, with a main size distribution of 50–200 nm and typical “cup-shaped” morphology. CD63 and CD9 proteins were highly expressed. PKH67 labeling confirmed effective uptake by keratinocytes.

In inflammation experiments, various doses of NSC-EVs significantly inhibited RAW264.7 macrophage activation and expression of inflammatory markers (IL-6, TNF-α, IL-1β, RANTES, MCP-1, iNOS, COX-2) and reduced NF-κB phosphorylation, demonstrating potent immunosuppressive effects.

2.4 In Vivo Efficacy in the AD Mouse Model

Topical application of NSC-CM and NSC-EVs significantly reduced the severity of AD-like lesions in mice, improved epidermal thickening and scabbing, and restored skin hair, with efficacy comparable to tacrolimus. Notably, histopathology showed a nearly 50% reduction in mast cell infiltration in the NSC-EVs group and significant thinning of epidermal thickness. PKH67 labeling confirmed that NSC-EVs could penetrate into both the epidermis and dermis, mainly distributed around hair follicles. This provides theoretical and experimental support for practical topical applications.

2.5 Proteomic Analysis

A total of 2,650 proteins were identified by LC-MS/MS, with the top 100 in abundance analyzed in depth. Interaction networks and GO enrichment indicated that some proteins (e.g., S100A8, SERPINA1, A2M) are closely related to immune regulation and anti-inflammation, while others (e.g., FN1, COL1A1, HSPG2, ITGB1) are involved in skin regeneration, wound healing, and ECM remodeling. In addition, plasma proteins and complement components (C3) were enriched, suggesting that NSC-EVs have dual functions in alleviating excessive inflammation and promoting skin tissue regeneration and repair.

IV. Conclusions and Significance

This study comprehensively demonstrates the potential therapeutic value of neural stem cell-derived extracellular vesicles and their conditioned media for atopic dermatitis. The primary conclusions are:

  • Both NSC-CM and NSC-EVs can effectively downregulate inflammatory factors in in vitro models, inhibit NF-κB signaling, reduce macrophage activation, and exhibit significant immunosuppressive effects.
  • Topical application of NSC-EVs in AD mice can significantly improve skin symptoms, inhibit mast cell inflammatory infiltration, promote epidermal barrier repair, and achieve efficacy comparable to first-line clinical drugs.
  • NSC-EVs are enriched in numerous bioactive proteins related to immune regulation and tissue regeneration. Through proteomics, the molecular basis of their effects is established, providing strong support for mechanistic studies and future development.

Scientifically, this study innovatively proposes the dual function of neural stem cell-derived extracellular vesicles in anti-inflammation and barrier repair in atopic dermatitis, offering a novel cell-free therapeutic approach. It provides an important reference for understanding new strategies for skin inflammation treatment and for the role of stem cell-derived exosomes.

In practical terms, NSC-EVs, as a form of nano-medicine that does not require live cell transplantation, offer higher purity, lower immunogenicity, and greater ease in topical formulation preparation. This provides a new expandable concept for future management of atopic dermatitis, complex chronic inflammatory skin diseases, and even broader applications in regenerative medicine.

V. Research Highlights and Innovations

  • Innovative Research Focus: This is the first systematic study of the role and mechanism of neural stem cell exosomes in atopic dermatitis, filling the research gap left by previous studies focused mainly on mesenchymal stem cells.
  • Outstanding Technical Approach: Emphasis on high-purity, patented Exo-i column for exosome isolation; use of cutting-edge techniques including TMT labeling LC-MS/MS and interactive data analysis with R; ensuring scientific and rigorous methodology.
  • Clear Targets and Mechanistic Integration: Simultaneously tracking the NF-κB pathway, inflammatory factors, histocytology, and multi-dimensional proteomic evidence, providing a clear mechanistic chain.
  • Broad Application Prospects: The potential for topical exosome formulations is clear, with favorable prospects for clinical translation, and offers a model for expanding the scope of nanomedicine.

VI. Additional Information

To ensure data quality, the study clearly describes statistical analysis methods (including multigroup ANOVA and appropriate multiple corrections), and all data or code can be requested from the corresponding author. The authors declare no potential conflicts of interest and have obtained relevant ethical approvals, ensuring the scientificity and compliance of the research.

The literature review also supplemented the progress and shortcomings of various types of stem cells and exosomes in AD and other diseases, with comparative analysis highlighting the distinctions and advantages of this study.

VII. Outlook and Implications

With rising incidences of chronic inflammatory and allergic skin diseases, novel, effective, and safe cell-free therapies are destined to become a hotspot. In-depth research into NSC-EVs and their proteomic systems will open new avenues for targeted immunomodulation and tissue regeneration. Future efforts can focus on their large-scale preparation, storage, and transdermal delivery (such as via microneedles, nano-carrier hydrogels, etc.), and integrate broader transcriptomic and functional experiments to promote translation to clinical application.

This research, rooted at the forefront of the field, with pioneering methods and multidimensional integration, provides an important reference for international studies and industrial expansion in dermatology and regenerative medicine.