Generation and characterization of MGE-derived GABAergic neurons from human pluripotent stem cells

越来越多的证据表明，GABA能中间神经元功能障碍与多种神经心理疾病有关。 这些疾病包括自闭症、精神分裂症、躁郁症和癫痫。最近成功地确定了可验证的 遗传风险因素与人类诱导多能性的技术和方法进展 以干细胞为基础的模型为我们提供了一个发展体外人类细胞模型的独特机会 进行机械调查。鉴于GABA能中间神经元的多样性，从平凡的 兴奋性-抑制性平衡失调的想法与不同的神经心理状况有关 从机制上理解不同中间神经元亚型对每种独特的病理生理学的贡献， 从人多能干细胞(HPSCs)中获得特定类型的GABA能细胞是非常必要的。然而，没有 到目前为止，这种方法在产生特定GABA能亚型的同质群体方面是有效的。 我们的长期目标是开发和使用人体模型系统来阐明分子和细胞机制-- GABA能神经元功能障碍相关的神经心理障碍。作为小白蛋白 (PV)和(SST)表达GABA能中间神经元起源于内侧神经节隆起 (MGE)已知在自闭症、精神分裂症和躁郁症患者中会受到影响，这里的目标是生成 并鉴定了人hPSCMGE来源的GABA能神经元。当前的中心假说 研究表明，使用外部模式因子和关键的谱系决定转录因子的组合， HPSC的分化可以被精确地引导来产生不同的GABA能细胞类型，这已经被 是根据以前的研究和我们基本上未公布的初步数据制定的。这一假说将 通过追求两个具体目标进行测试：1)通过Sequen-Sequen引导人类神经前体细胞规范 神经元来源和亚型受限关键因子的表达；2)转录特异性人MGE 转化为GABA能神经元亚型的祖细胞。在第一个目标下，我们将模拟基因级联 在发育过程中通过异位表达空间和时间-来指定GABA能中间神经元细胞的命运 HPSCs来源的前脑祖细胞中的部分特异性转录因子。在第二个目标下，我们将 通过异位表达，转录指定使用不同方法产生的MGE祖细胞 表达PV和SST的GABA能中间神经元特异性因子。将使用多学科方法来 描述所产生的神经元的同一性、异质性、成熟阶段和功能。一起应用， 这些目标将使我们能够使用以前建立的方法作为起点，并开发新的方法 结合外在因素和内在转录规范来产生特定的GABA能细胞类型 来自人类的PSCs。鉴于表达PV和SST的GABA能中间神经元功能障碍的高发 在神经心理障碍方面，这项工作将为未来具有积极意义的研究提供基本工具。 有必要阐明这些复杂疾病背后的关键分子和细胞机制。

Increasing evidence implicates dysfunction of GABAergic interneurons in a host of neuropsychological condi- tions, including autism, schizophrenia, bipolar disorder, and epilepsy. Recent success in identifying veritable genetic risk factors together with the technological and methodological advances in human induced pluripotent stem (iPS) cell-based models have rendered us a unique opportunity to develop in vitro human cellular models for mechanistic investigations. Given the diversity of GABAergic interneurons, to move from the commonplace idea that disturbed excitatory-inhibitory balance is associated with various neuropsychological conditions toward a mechanistic understanding of the contribution of different interneuron subtypes to each unique pathophysiology, it is essential to derive specific GABAergic cell types from human pluripotent stem cells (hPSCs). However, no approach to date has been effective in generating homogeneous populations of specific GABAergic subtypes. Our long-term goal is to develop and use human model systems to elucidate the molecular and cellular mecha- nisms underlying GABAergic neuron dysfunction-associated neuropsychological disorders. As parvalbumin (PV)- and somatostatin (SST)-expressing GABAergic interneurons originated from medial ganglionic eminence (MGE) are known to be affected in autism, schizophrenia, and bipolar patients, the objective here is to generate and characterize MGE-derived GABAergic neurons from human hPSCs. The central hypothesis of the current study is that using a combination of external patterning factors and key lineage-determining transcription factors, the differentiation of hPSCs can be precisely directed to produce distinct GABAergic cell types, which has been formulated on the basis of previous studies and our largely unpublished Preliminary Data. The hypothesis will be tested by pursuing two specific aims: 1) To guide human neural progenitor cell specification through sequen- tial expression of neuron origin- and subtype-restricted key factors; 2) To transcriptionally specify human MGE progenitors into GABAergic neuron subtypes. Under the first aim, we will mimic the genetic cascade that takes place during development to specify GABAergic interneuron cell fate by ectopic expression of spatial and tem- poral specific transcription factors in forebrain progenitors derived from hPSCs. Under the second aim, we will transcriptionally specify MGE progenitor cells generated using different methods through ectopic expression of PV- and SST-expressing GABAergic interneuron specific factors. A multidisciplinary approach will be used to characterize the identity, heterogeneity, maturation stage and function of neurons generated. Applied together, these aims will allow us to use previously established methods as a starting point and develop new approaches that combine extrinsic factors and intrinsic transcriptional specifications to generate specific GABAergic cell types from human PSCs. Given the high prevalence of PV- and SST-expressing GABAergic interneuron dysfunction in neuropsychological disorders, this work will provide the fundamental tool for future studies that have the po- tential to shed light on the key molecular and cellular mechanisms underlying these complex disease conditions.