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Abstract As current treatments for Alzheimer's disease (AD) lack disease‐modifying interventions, novel therapies capable of restraining AD progression and maintaining better brain function have great significance. Anti‐inflammatory extracellular vesicles (EVs) derived from human induced pluripotent stem cell (hiPSC)‐derived neural stem cells (NSCs) hold promise as a disease‐modifying biologic for AD. This study directly addressed this issue by examining the effects of intranasal (IN) administrations of hiPSC‐NSC‐EVs in 3‐month‐old 5xFAD mice. IN administered hiPSC‐NSC‐EVs incorporated into microglia, including plaque‐associated microglia, and encountered astrocyte soma and processes in the brain. Single‐cell RNA sequencing revealed transcriptomic changes indicative of diminished activation of microglia and astrocytes. Multiple genes linked to disease‐associated microglia, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3)‐inflammasome and interferon‐1 (IFN‐1) signalling displayed reduced expression in microglia. Adding hiPSC‐NSC‐EVs to cultured human microglia challenged with amyloid‐beta oligomers resulted in similar effects. Astrocytes also displayed reduced expression of genes linked to IFN‐1 and interleukin‐6 signalling. Furthermore, the modulatory effects of hiPSC‐NSC‐EVs on microglia in the hippocampus persisted 2 months post‐EV treatment without impacting their phagocytosis function. Such effects were evidenced by reductions in microglial clusters and inflammasome complexes, concentrations of mediators, and end products of NLRP3 inflammasome activation, the expression of genes and/or proteins involved in the activation of p38/mitogen‐activated protein kinase and IFN‐1 signalling, and unaltered phagocytosis function. The extent of astrocyte hypertrophy, amyloid‐beta plaques, and p‐tau were also reduced in the hippocampus. Such modulatory effects of hiPSC‐NSC‐EVs also led to better cognitive and mood function. Thus, early hiPSC‐NSC‐EV intervention in AD can maintain better brain function by reducing adverse neuroinflammatory signalling cascades, amyloid‐beta plaque load, and p‐tau. These results reflect the first demonstration of the efficacy of hiPSC‐NSC‐EVs to restrain neuroinflammatory signalling cascades in an AD model by inducing transcriptomic changes in activated microglia and reactive astrocytes.

Grafting of neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise for brain repair after injury or disease, but safety issues have hindered their clinical application. Employing nano-sized extracellular vesicles (EVs) derived from hiPSC-NSCs appears to be a safer alternative because they likely have similar neuroreparative properties as NSCs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. However, reliable methods for isolation, characterization and testing the biological properties of EVs are critically needed for translation. We investigated signatures of miRNAs and proteins and the biological activity of EVs, isolated from hiPSC-NSCs through a combination of anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC). AEC and SEC facilitated the isolation of EVs with intact ultrastructure and expressing CD9, CD63, CD81, ALIX and TSG 101. Small RNA sequencing, proteomic analysis, pathway analysis and validation of select miRNAs and proteins revealed that EVs were enriched with miRNAs and proteins involved in neuroprotective, anti-apoptotic, antioxidant, anti-inflammatory, blood-brain barrier repairing, neurogenic and Aβ reducing activities. Besides, EVs comprised miRNAs and/or proteins capable of promoting synaptogenesis, synaptic plasticity and better cognitive function. Investigations using an in vitro macrophage assay and a mouse model of status epilepticus confirmed the anti-inflammatory activity of EVs. Furthermore, the intranasal administration of EVs resulted in the incorporation of EVs by neurons, microglia and astrocytes in virtually all adult rat and mouse brain regions, and enhancement of hippocampal neurogenesis. Thus, biologically active EVs containing miRNAs and proteins relevant to brain repair could be isolated from hiPSC-NSC cultures, making them a suitable biologic for treating neurodegenerative disorders.