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Abstract：
Generation of human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) offers an unprecedented approach to modeling movement disorders such as dystonia and amyotrophic lateral sclerosis. However, achieving survival poses a significant challenge when culturing induced MNs, especially when aiming to reach late maturation stages. Utilizing hiPSC-derived motor neurons and primary mouse astrocytes, we assembled two types of coculture systems: direct coculturing of neurons with astrocytes and indirect coculture using culture inserts that physically separate neurons and astrocytes. Both systems significantly enhance neuron survival. Compared with these two systems, no significant differences in neurodevelopment, maturation, and survival within 3 weeks, allowing to prepare neurons at maturation stages. Using the indirect coculture system, we obtained highly pure MNs at the late mature stage from hiPSCs. Transcriptomic studies of hiPSC-derived MNs showed a typical neurodevelopmental switch in gene expression from the early immature stage to late maturation stages. Mature genes associated with neurodevelopment and synaptogenesis are highly enriched in MNs at late stages, demonstrating that these neurons achieve maturation. This study introduces a novel tool for the preparation of highly pure hiPSC-derived neurons, enabling the determination of neurological disease pathogenesis in neurons at late disease onset stages through biochemical approaches, which typically necessitate highly pure neurons. This advancement is particularly significant in modeling age-related neurodegeneration.
摘要：
人类诱导多能干细胞（hiPSC）衍生的运动神经元（MN）的产生提供了一种前所未有的方法来模拟运动障碍，例如肌张力障碍和肌萎缩性侧索硬化症。然而,在培养诱导的MNs时，实现存活构成了重大挑战，特别是当目标是达到成熟后期阶段时。利用hiPSC衍生的运动神经元和原代小鼠星形胶质细胞，我们组装了两种类型的共培养系统：神经元与星形胶质细胞的直接共培养，和使用物理分离神经元和星形胶质细胞的培养插入物的间接共培养。两个系统都显著增强神经元存活。与这两个系统相比，神经发育没有显著差异，成熟,在3周内存活，允许在成熟期准备神经元。使用间接共培养系统，我们在成熟后期从hiPSC获得了高纯度的MNs。hiPSC衍生的MN的转录组学研究显示，从早期未成熟阶段到晚期成熟阶段，基因表达存在典型的神经发育转换。与神经发育和突触发生相关的成熟基因在晚期阶段高度富集在MNs中。证明这些神经元实现了成熟。这项研究介绍了一种用于制备高纯度hiPSC衍生神经元的新工具，能够通过生化方法在疾病发作后期确定神经疾病的发病机理，这通常需要高纯度的神经元。这种进步在建模与年龄相关的神经变性中特别重要。意义陈述实现存活对长期神经细胞培养提出了重大挑战。利用hiPSC衍生的运动神经元和原代小鼠星形胶质细胞，我们建立了一个间接共培养系统，使用培养插入物物理分离神经元和星形胶质细胞，从而促进神经元成熟。转录组学研究揭示了从早期未成熟阶段到晚期成熟阶段的基因表达中典型的神经发育转换,表明用培养插入物制备的神经元的高质量和成熟。这项研究介绍了一种用于制备高纯度hiPSC衍生神经元的新工具，能够通过生化方法在疾病发作后期确定神经疾病的发病机理，这通常需要高纯度的神经元。这种进步在建模与年龄相关的神经变性中特别重要。
