Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors.
Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors.
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DOI：
10.1002/cpz1.141
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发表时间：
2021-06
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影响因子：
--
通讯作者：
Sproul A
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文献类型：
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作者：
Song S;Ashok A;Williams D;Kaufman M;Duff K;Sproul A
为了了解细胞类型在脑功能和对疾病病理的易感性中的特定作用，必须产生分离的人类神经元亚型群体。在这里，我们描述了一种利用神经源性转录因子（tf） Ngn2和Ascl1和Dlx2的组合，从人多能干细胞（hPSCs）平行生成皮质谷氨酸能（兴奋性）和gaba能（抑制性）神经元的方案。与大多数使用短暂慢病毒感染的神经转分化方案相反，该方案利用稳定的hPSC系与强力霉素诱导的tf，然后可以通过添加强力霉素和神经介质进行神经分化。首先，我们提出了一种从哺乳动物培养的细胞中产生慢病毒的方法，并建立了含有tfs的稳定细胞系（基本方案1），然后我们描述了从已建立的hPSCs中获得的单层兴奋性和抑制性神经元分化（基本方案2）。由此产生的神经元可复制地表现出与人类皮质神经元一致的特性，包括预期的形态、谷氨酸能和gaba能基因的表达以及功能特性。我们的方法能够扩展和快速生产适合以亚型特异性方式建模人类大脑疾病和检查差异细胞脆弱性的人类神经元。基本协议1：慢病毒的产生和稳定hPSC细胞系的创建基本协议2:EX-和in -神经元分化支持协议1：扩展和维持hPSCs支持协议2：验证EX-和in -神经元的实验方法
It is essential to generate isolated populations of human neuronal subtypes in order to understand cell-type specific roles in brain function and susceptibility to disease pathology. Here we describe a protocol for in-parallel generation of cortical glutamatergic (excitatory) and GABAergic (inhibitory) neurons from human pluripotent stem cells (hPSCs) by using the neurogenic transcription factors (TFs) Ngn2, and the combination of Ascl1 and Dlx2, respectively. In contrast to the majority of neural transdifferentiation protocols which use transient lentiviral infection, this protocol utilizes stable hPSC lines with doxycycline-inducible TFs, which can then be neuronally differentiated by addition of doxycycline and neural media. First, we present a method to generate lentivirus from mammalian cultured cells and establish TFs-incorporated stable cell lines (Basic Protocol 1), and then we describe a monolayer excitatory and inhibitory neuronal differentiation derived from the established hPSCs (Basic Protocol 2). The resulting neurons reproducibly exhibit properties consistent with human cortical neurons, including the expected morphologies, expression of glutamatergic and GABAergic genes, and functional properties. Our approach enables the scalable and rapid production of human neurons suitable for modeling human brain diseases in a subtype-specific manner and examination of differential cellular vulnerability. Basic Protocol 1: LENTIVIRUS PRODUCTION AND CREATION OF STABLE hPSC LINES Basic Protocol 2: EX-, and IN-neuron differentiation Support Protocol 1: Expansion and maintenance of hPSCs Support Protocol 2: Experimental methods for validation of EX- and IN-neurons