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.

从人类诱导的多能干细胞（hiPSC）衍生的神经干细胞（NSC）的嫁接已显示出在受伤或患病后进行脑修复的希望，但安全性问题阻碍了它们的临床应用。使用源自hiPSC-NSC的纳米级细胞外囊泡（EV）似乎是一种更安全的选择，因为它们可能具有与NSC相似的神经修复特性，并且适合作为自体或异源现成产品进行非侵入性给药。然而，翻译的关键是需要可靠的方法来隔离，表征和测试EV的生物学特性。我们调查了通过结合阴离子交换色谱（AEC）和尺寸排阻色谱（SEC）从hiPSC-NSC中分离出的miRNA和蛋白质的特征以及EV的生物学活性。AEC和SEC促进了具有完整超微结构并表达CD9，CD63，CD81，ALIX和TSG 101的电动汽车的分离。小分子RNA测序，蛋白质组学分析，通路分析和选定的miRNA和蛋白质的验证表明，电动汽车富含miRNA和涉及的蛋白质具有神经保护，抗凋亡，抗氧化剂，抗炎，血脑屏障修复，神经源性和Aβ还原活性。此外，电动汽车包含能够促进突触发生，突触可塑性和更好的认知功能的miRNA和/或蛋白质。调查使用 某些miRNA和蛋白质的信号通路分析和验证表明，EV富含与神经保护，抗凋亡，抗氧化剂，抗炎，血脑屏障修复，神经源性和Aβ还原活性有关的miRNA和蛋白质。此外，电动汽车包含能够促进突触发生，突触可塑性和更好的认知功能的miRNA和/或蛋白质。调查使用 某些miRNA和蛋白质的信号通路分析和验证表明，EV富含与神经保护，抗凋亡，抗氧化剂，抗炎，血脑屏障修复，神经源性和Aβ还原活性有关的miRNA和蛋白质。此外，电动汽车包含能够促进突触发生，突触可塑性和更好的认知功能的miRNA和/或蛋白质。调查使用体外巨噬细胞测定和癫痫持续状态小鼠模型证实了电动汽车的抗炎活性。此外，鼻内施用EV导致几乎所有成年大鼠和小鼠脑区域中神经元，小胶质细胞和星形胶质细胞掺入EV，并增强海马神经发生。因此，可以从hiPSC-NSC培养物中分离出含有与大脑修复有关的miRNA和蛋白质的具有生物活性的EV，使其成为治疗神经退行性疾病的合适生物制剂。