Ken & Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
Sci Rep. 2021 Nov 15;11(1):22257. doi: 10.1038/s41598-021-01366-4.
During cellular specification, transcription factors orchestrate cellular decisions through gene regulation. By hijacking these transcriptional networks, human pluripotent stem cells (hPSCs) can be specialized into neurons with different molecular identities for the purposes of regenerative medicine and disease modeling. However, molecular fine tuning cell types to match their in vivo counterparts remains a challenge. Directing cell fates often result in blended or incomplete neuron identities. A better understanding of hPSC to neuron gene regulation is needed. Here, we used single cell RNA sequencing to resolve some of these graded molecular identities during human neurogenesis from hPSCs. Differentiation platforms were established to model neural induction from stem cells, and we characterized these differentiated cell types by 10x single cell RNA sequencing. Using single cell trajectory and co-expression analyses, we identified a co-regulated transcription factor module expressing achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1) and neuronal differentiation 1 (NEUROD1). We then tested the function of these transcription factors in neuron subtype differentiation by gene knockout in a novel human system that reports the expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. ASCL1 was identified as a necessary transcription factor for regulating dopaminergic neurotransmitter selection.
在细胞特化过程中,转录因子通过基因调控来协调细胞决策。通过劫持这些转录网络,人类多能干细胞 (hPSC) 可以被特化为具有不同分子特征的神经元,用于再生医学和疾病建模。然而,将细胞类型进行分子微调以匹配其体内对应物仍然是一个挑战。定向细胞命运通常会导致混合或不完全的神经元身份。需要更好地了解 hPSC 到神经元的基因调控。在这里,我们使用单细胞 RNA 测序来解决人类神经发生过程中从 hPSC 到神经元的一些这些渐变分子特征。建立了分化平台来模拟干细胞的神经诱导,我们通过 10x 单细胞 RNA 测序来表征这些分化的细胞类型。通过单细胞轨迹和共表达分析,我们鉴定了一个共调控的转录因子模块,表达 achaete-scute 家族基本螺旋-环-螺旋转录因子 1 (ASCL1) 和神经元分化 1 (NEUROD1)。然后,我们通过在一种新型的人类系统中敲除这些转录因子来测试它们在神经元亚型分化中的功能,该系统报告酪氨酸羟化酶 (TH) 的表达,TH 是多巴胺合成中的限速酶。ASCL1 被鉴定为调节多巴胺能神经递质选择所必需的转录因子。