Defining a transcriptional periodic table of the human brain using reprogramming

人类大脑包含不同的神经细胞类型，这些神经细胞类型不同地负责不同的方面， 人类行为、认知和神经系统疾病。DNA测序的进展提供了新的见解 通过提供增加疾病风险或与疾病相关的基因列表， 行为和认知特性在个体之间存在差异。然而，由于缺乏可用的人力 神经元亚型和我们对哪些基因在不同的人类神经元中表达的有限理解， 开发这些不断增长的基因组资源的主要障碍。克服这一障碍的一种方法是直接使用 通过瞬时表达转录因子（TF）进行重编程以在体外产生诱导的神经元细胞 在成纤维细胞中。直接重编程产生的诱导神经元与内源性神经元具有许多共同特征， 神经元，包括特征形态，配体诱发的突触反应和特征模式 的基因表达。因此，重编程提供了一种新的工具来识别转录回路， 神经元身份的独特特征。我们和其他人已经使用候选基因的方法来设计 诱导的神经元在功能上模仿特征良好的神经元亚型，如外周感觉神经元。 我的实验室最近制造出了一种能够感知疼痛和瘙痒的神经元。这些研究让我们假设， 直接重编程涉及保守的转录回路，类似于那些积极维持 内源性神经元中的神经元亚型身份。这一假设预测，应该有可能确定 多种TF组合诱导不同神经元亚型的不同特征。为了验证这一假设 并建立一个系统的方法来产生和分类人类神经元亚型，我们将进行一个 无偏筛选新的TF组合，可以在体外诱导人类神经元亚型。然后我们将 从转录、形态和功能上表征诱导的神经元。我们非常适合 进行这项研究，因为我们最近进行了约600个TF对的试点筛选，并确定了70多个 从小鼠成纤维细胞产生候选诱导神经元的新对。基因表达谱和 这些细胞的功能分析证实它们表现出广泛的亚型多样性。因此，通过使用 无偏筛选来定义可以在成纤维细胞中诱导神经元身份的人TF集，我们将鉴定 在体外产生具有确定功能特性的人类神经元细胞类型的新方法， 转录程序和细胞特性的数据库，这些程序和细胞特性是由不同的 TF的集合。这些研究将对我们理解人类神经元的基础生物学产生影响。 多样性，并将提供概念和实用的工具，使神经科学研究人员产生多样化的 用于研究和转化应用的人类诱导神经元的亚型。

The human brain contains diverse neural cell types that are differentially responsible for distinct aspects of human behavior, cognition and neurologic disease. Advances in DNA sequencing are providing new insight into human specific neurobiology by providing lists of genes that increase risk for disease or correlate with behaviors and cognitive properties that differ between individuals. However, the lack of available human neuronal subtypes and our limited understanding of which genes are expressed in different human neurons are major barriers to exploiting these growing genomic resources. One way to overcome this barrier is to use direct reprogramming to produce induced neuronal cells in vitro, by transiently expressing transcription factors (TFs) in fibroblasts. Direct reprogramming produces induced neurons that share many features with endogenous neurons, including characteristic morphologies, ligand-evoked synaptic responses and characteristic patterns of gene expression. Reprogramming therefore offers a new tool to identify transcriptional circuits that establish distinct features of neuronal identity. We, and others, have used candidate gene approaches to engineer induced neurons that functionally mimic well-characterized neuronal subtypes, such as the peripheral sensory neurons that detect pain and itch produced recently by my laboratory. These studies led us to hypothesize that direct reprogramming engages conserved transcriptional circuits similar to those that actively maintain neuronal subtype identity in endogenous neurons. This hypothesis predicts that it should be possible to identify multiple TF combinations that induce distinct features of different neuronal subtypes. To test this hypothesis and establish a systematic method to produce and classify human neuronal subtypes, we will conduct an unbiased screen for new TF combinations that can induce human neuronal subtypes in vitro. We will then characterize the induced neurons transcriptionally, morphologically and functionally. We are well suited to perform this study because we recently conducted a pilot screen of ~600 TF pairs and identified more than 70 new pairs that produce candidate induced neurons from mouse fibroblasts. Gene expression profiling and functional analyses of these cells confirm that they exhibit extensive subtype diversity. Therefore, by using unbiased screens to define sets of human TFs that can induce neuronal identity in fibroblasts, we will identify new methods to produce human neuronal cell types with defined functional properties in vitro and establish a database of transcriptional programs and cellular properties that emerge from transient expression of different sets of TFs. These studies will have impact on our understanding of the basic biology of human neuronal diversity and will provide conceptual and practical tools to enable neuroscience researchers to produce diverse subtypes of human induced neurons for research and translational applications.