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

Abstract

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-theshelf 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 antiinflammatory 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.

Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

“…Three similar studies to ours in rodent brain tissue found that within 2 and 6 h after intranasal administration, EVs were widely distributed into the somatosensory and prefrontal cortex, amygdala, dentate gyrus, and cerebellum [65,66]. Similar to our findings, EVs were in neurons, microglia, and astrocytes [66], and completely cleared from brain within 24 h [67].…”

Insights Into Exosomal Non-Coding RNAs Sorting Mechanism and Clinical Application

“…Exosomal miRNAs and proteins isolated from hiPSC-NSC cultures have many functions, such as neuroprotection and anti-inflammation. The intranasal administration of exosomes can be absorbed by a variety of nerve cells, which is beneficial for brain repair after injury or disease ( 139 ). A separate study showed that secreted exosomes coated with miR-146a-5p from MSCs relieved Group 2 innate lymphoid cells (ILC2s) in innate airway inflammation, showing significant advantages of low immunogenicity and high biosafety ( 140 ).…”

Ancient Evolutionary Origin and Properties of Universally Produced Natural Exosomes Contribute to Their Therapeutic Superiority Compared to Artificial Nanoparticles

“…Exosomes have a natural ability to cross the normal, unperturbed blood–brain vascular barrier [ 95 , 96 , 97 ] by transcytosis [ 95 , 224 ], thus enabling the systemic treatment of CNS-inflammatory disorders [ 238 ] and possibly cancers [ 239 ], even when administered by the nasal route [ 238 , 240 , 241 , 242 ].…”

“…There is a revolutionary ability to administer exosomes via the nasal route. These exosomes then traffic around cranial nerve I fibers for smell, via the cribriform plate of the skull, to transit into the brain for the treatment of neuro-inflammatory conditions [ 246 ], like models of viral encephalitis and multiple sclerosis [ 246 ], brain injury [ 238 , 241 ], and even attempts to treat a mouse model of autism [ 242 ] and prevent brain damage from epilepsy [ 240 ]; interestingly, even when given via a nasal spray is sufficient [ 241 ]. The further exploitation of the exosome nasal route of administration may treat brain tumors [ 235 ] and spinal cord injury [ 239 ] or can be directed at airway inflammation [ 247 ], chronic rhinosinusitis [ 248 ], and possibly COVID-19 lung infections.…”