Extracellular vesicles from human iPSC‐derived neural stem cells: miRNA and protein signatures, and anti‐inflammatory and neurogenic properties

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 <i>in vitro</i> 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.