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: 8545242
• 负责人: TIMOTHY J PETROS
• 金额: $ 5.41万
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8545242
• 关键词: Advanced Development; Autistic Disorder; Brain; Cell Therapy; Cell Transplantation; Cells; Cerebral cortex; Characteristics; Collection; Derivation procedure; Development; Disabled Persons; Disease; Disease model; ES Cell Line; Embryo; Engineering; Epilepsy; Equilibrium; Erinaceidae; Etiology; Functional disorder; Future; Gene Expression Profile; Generations; Genes; Genetic Determinism; Interneurons; Location; Medial; Mental disorders; Mitotic; Molecular Genetics; Mus; Neonatal; Neurons; Parvalbumins; Physiological; Play; Production; Property; Prosencephalon; Protocols documentation; RNA; Reporter; Research; Research Personnel; Role; Schizophrenia; Somatostatin; Source; Specific qualifier value; Stem cells; Subgroup; Sum; Synapses; Techniques; Telencephalon; Therapeutic; Time; Training; Transcript; Transplantation; base; career; cell type; embryonic stem cell; genetic manipulation; handicapping condition; in vivo; innovation; molecular marker; nervous system disorder; neurochemistry; neurodevelopment; neurotransmission; novel; postnatal; pre-doctoral; progenitor; promoter; research study; smoothened signaling pathway; stem; stem cell differentiation; tool; transcription factor; transcriptome sequencing

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抑制的主要来源，通过平衡皮层回路中的兴奋，在大脑功能的几乎各个方面发挥着至关重要的作用。中间神经元约占皮质神经元的 20%，根据其神经化学标记、连接性和生理特性分为不同的亚型。然而，人们对产生不同中间神经元亚型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。缺乏有效的中间神经元的产生、收集和选择机制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其作为细胞疗法的潜力的能力。目前从胚胎干 (ES) 细胞中获取中间神经元的方案受到所产生的中间神经元比例较小以及表达生长抑素 (Sst+) 的中间神经元相对于其他亚组的偏向产生的限制。在这项提案中，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，是中间神经元亚型规范所必需的。我们正在追求的一项策略是在 ES 细胞中诱导表达 Nkx2.1，该细胞还含有用于中间神经元命运的有丝分裂后报告基因 (Lhx6:GFP)。另一种策略是利用 Nkx2.1:mChery;Lhx6:GFP ES 系，它使我们能够特异性分离 Nkx2.1+ 祖细胞和新有丝分裂后的 Lhx6+ 细胞，这有助于富集 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，以确认这些细胞在体内表达功能性中间神经元的神经化学和电生理学特性。在建立产生高比例 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+ 细胞收集 RNA，并进行 RNA-seq 分析来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会提高我们生产 ES 细胞衍生的中间神经元的能力，特别是生产特定亚型中间神经元的能力。这些结果将极大地帮助中间神经元发育的研究，并提高我们使用中间神经元作为基于细胞的疾病治疗方法的能力。