Intranasal Delivery of Hypoxia-Primed Wharton’s Jelly MSC-Derived sEVs Reprograms Neuroimmune Signalling and Ameliorates Behavioural Deficits in a Valproic Acid–Induced Mouse Model of Autism Spectrum Disorder

Background

Beyond disrupted neurodevelopment, autism spectrum disorder (ASD) encompasses progressive neurodegenerative alterations that remain refractory to standard of care behavioral therapies, highlighting the need for biologically targeted adjunctive interventions. Although, Mesenchymal stem cell (MSC) derived small extracellular vesicles (sEVs) have emerged as potent neuroprotective agents; however, the optimal MSC tissue source and strategies enhancing their efficacy for clinical translation remain undefined. Hypoxic preconditioning potentiates their therapeutic effects, however its specific impact on ASD-related impairments is yet to be established. This study conducts a head to head evaluation of sEVs from bone marrow (BM) and Whartons jelly (WJ) MSCs under normoxic and hypoxic conditions to delineate their comparative neuroregenerative efficacy in ASD.

Methods

We investigated the neuroprotective efficacy of sEVs derived from human tissue-specific mesenchymal stem cells (BM/WJ) cultured under normoxic (21% O₂) and hypoxic (1% O₂) conditions. In vitro assays evaluated sEV uptake, cell proliferation, neurodifferentiation, mitochondrial health, and immunomodulatory effects using neural stem cell (C17.2) and microglial (N9) cell lines. For in vivo studies, ASD-like phenotypes were induced in C57BL/6 mice via prenatal exposure to valproic acid (600 mg/kg), followed by the biodistribution of fluorescently labeled hypoxia-primed WJ MSCs sEVs ( WJ-H-sEVs) following intranasal administration was assessed using IVIS imaging. Behavioural outcomes after intranasal treatment were evaluated. Additionally, post-mortem brain tissues were analyzed for neuroinflammation, oxidative stress, synaptic integrity, and downstream signalling pathways using immunofluorescence, Western blotting, and qRT-PCR, while systemic cytokine pro-inflammatory and anti-inflammatory levels were quantified by ELISA. Mechanistic involvement of Nrf2 and NF-κB signaling pathways was examined using pharmacological inhibitors.

Results

Comparative analyses revealed that hypoxic priming significantly enhanced sEV yield and enriched neuroprotective miRNAs, with WJ-H-sEVs exhibiting elevated levels of miR-125b-5p, miR-21a-5p, and miR-145a-5p. Consistent with this enrichment, WJ-H-sEVs demonstrated superior cellular uptake as observed in neural stem cells and microglia, enhanced neurodifferentiation, robust antioxidant activity, effective immunomodulation, and improved mitochondrial health, thereby outperforming their normoxic and BM-derived counterparts. Intranasally administered WJ-H-sEVs efficiently localized to the brain within 12 h and significantly improved anxiety-like behavior, recognition memory, and spatial learning in ASD mice. These effects were accompanied by reduced neuroinflammation and oxidative damage, enhanced neuronal survival, decreased serum IL-6 levels, and increased IL-10 and TGF-β. Mechanistically, therapeutic benefits were mediated through activation of the Nrf2 antioxidant pathway and suppression of NF-κB and JAK-STAT signaling.

Conclusion

In this study, hypoxia emerges as a clinically exploitable priming strategy to enhance the neuroregenerative performance of tissue-specific MSC-derived sEVs, with potential applicability across MSC sources and other neurological disorders. Moreover, intranasal delivery of WJ-H-sEVs demonstrates translational feasibility, reinforcing their potential as an adjunctive, cell-free, pediatric-friendly neurotherapeutic modality for ASD management.

Graphical Abstract