Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.

通过神经原 2 ( NGN2 ) 过表达从诱导多能干细胞 (iPSC) 工程改造的人类神经元被广泛用于研究神经元分化机制和模拟神经系统疾病。然而，出现的神经元的分化路径和异质性尚未得到充分探索。在这里，我们使用单细胞转录组学来剖析NGN2期间出现的细胞状态从多能性到神经元功能成熟的时间过程中过度表达。我们发现产生的神经元类型存在显着的分子异质性，至少有两个群体表达与周围神经系统神经元相关的基因。在来自不同个体的多个 iPSC 克隆和系中观察到神经元异质性。我们发现神经元命运获取对NGN2表达水平和 NGN2 强制表达的持续时间很敏感。我们的数据表明，NGN2剂量可以调节神经元命运的获得，并且NGN2 -iN异质性可以混淆对神经元类型敏感的结果。