Generation of interneurons derived from ES cells using inducible expression of tr

使用 tr 的诱导表达产生源自 ES 细胞的中间神经元

• 批准号: 8501976
• 负责人: TIMOTHY J PETROS
• 金额: $ 0.78万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-13 至 2014-09-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8501976
• 关键词: Generation; interneurons; derived; ES; cells

DESCRIPTION (provided by applicant): Forebrain GABAergic interneurons are the primary source of inhibition in the cerebral cortex and play crucial roles in nearly every aspect of brain function by balancing excitation in cortical circuits. Interneurons comprise approximately 20% of cortical neurons and are divided into different subtypes based on their neurochemical markers, connectivity and physiological properties. However, very little is known about the genetic and molecular mechanisms that give rise to different subtypes of interneuron. The abnormal development and function of cortical interneurons has been implicated in the pathobiology of several major neurological and psychiatric disorders, including schizophrenia, autism, epilepsy, and numerous other diseases. The lack of an efficient mechanism for the production, collection and selection of interneurons has greatly hindered our ability to study both the role of interneurons in disease etiologies, as well as analyze their potential as cell based therapies. The current protocol to derive interneurons from embryonic stem (ES) cells is handicapped by the small percentage of interneurons produced and the biased production of somatostatin- expressing (Sst+) interneurons over other subgroups. In this proposal, we hope to enhance the overall production of ES-derived interneurons and specifically enrich for parvalbumin-expressing (PV+) interneurons. Forced expression of key fate-determining transcription factors can direct the differentiation of stem cells into specific cell types. The transcription factor Nkx2.1 is expressed by cortical interneuron progenitors and is required for the specification of interneuron subtypes. One strategy we are pursuing is to inducibly express Nkx2.1 in ES cells that also contain a post-mitotic reporter for interneuron fate (Lhx6:GFP). Another strategy is to utilize an Nkx2.1:mChery;Lhx6:GFP ES line that allows us to specifically isolate Nkx2.1+ progenitor cells and newly-postmitotic Lhx6+ cels, which could help enrich for PV+ interneurons. We will collect and transplant GFP+ cells into the cortex of neonatal mice to confirm that these cells express neurochemical and electrophysiological properties characteristic of functional interneurons in vivo. After establishing distinct protocols that produce a high percentage of Sst+ and PV+ interneurons, we will collect RNA from GFP+ cells obtained from these different conditions and perform RNA-seq analysis to characterize the transcriptome and identify novel subgroup-specific genes that determine interneuron fate. In sum, the experiments outlined in this proposal should increase our ability to produce ES cell-derived interneurons, and in particular, production of specific subtypes of interneurons. These results will significantly aid the study of interneuron development and advance our capabilities to use interneurons as cell-based therapies for the treatment of disease.

描述（由申请人提供）：前脑GABA能中间神经元是大脑皮层中抑制的主要来源，并通过平衡皮层回路中的兴奋在大脑功能的几乎每个方面发挥关键作用。中间神经元包括大约20%的皮质神经元，并根据其神经化学标记物、连接性和生理特性分为不同的亚型。然而，人们对产生不同亚型的中间神经元的遗传和分子机制知之甚少。皮质中间神经元的异常发育和功能与几种主要神经和精神疾病的病理生物学有关，包括精神分裂症、自闭症、癫痫和许多其他疾病。缺乏有效的机制来产生、收集和选择中间神经元，极大地阻碍了我们研究中间神经元在疾病病因学中的作用以及分析它们作为基于细胞的疗法的潜力的能力。目前从胚胎干（ES）细胞衍生中间神经元的方案受到所产生的中间神经元的小百分比和相对于其他亚群的生长抑素表达（Sst+）中间神经元的偏向性产生的阻碍。在这个提议中，我们希望提高ES衍生的中间神经元的整体产量，并特别富集表达小白蛋白（PV+）的中间神经元。 关键的命运决定转录因子的强制表达可以指导干细胞分化为特定的细胞类型。转录因子Nkx2.1由皮质中间神经元祖细胞表达，并且是中间神经元亚型的特化所需的。我们正在追求的一个策略是在ES细胞中诱导表达Nkx2.1，该ES细胞还含有用于中间神经元命运的有丝分裂后报告基因（Lhx 6：GFP）。另一种策略是利用Nkx2.1：mChery; Lhx 6：GFP ES系，其允许我们特异性分离Nkx2.1+祖细胞和新的有丝分裂后Lhx 6+细胞，这可以帮助富集PV+中间神经元。我们将收集GFP+细胞并将其移植到新生小鼠的皮质中，以确认这些细胞表达体内功能性中间神经元的神经化学和电生理特性。在建立了产生高比例Sst+和PV+中间神经元的不同方案后，我们将从这些不同条件下获得的GFP+细胞中收集RNA，并进行RNA-seq分析以表征转录组并鉴定决定中间神经元命运的新亚组特异性基因。 总之，本提案中概述的实验应该增加我们产生ES细胞衍生的中间神经元的能力，特别是产生特定亚型的中间神经元。这些结果将大大有助于研究中间神经元的发育，并提高我们使用中间神经元作为治疗疾病的细胞疗法的能力。