Mapping the Developing Human Neocortex by Massively Parallel Single Cell Analysis

 描述（由申请人提供）：该提案旨在创建发育中的人类新皮层的单细胞分辨率图。我们建议确定产生大脑皮层的神经干细胞和祖细胞的不同亚型的数量，然后跟踪它们产生的新生神经元的发育轨迹，以了解最终形成成人皮层的皮层神经元的多样性。我们计划一种新的方法来解决这个问题，通过整合调查的单细胞基因表达和生理学在人类皮层细胞从多个大脑区域在一系列的发展阶段。与Fluidigm公司合作，我们开发了创新的策略，使用微流体技术，细胞条形码和时间推移显微镜对原代人类皮质细胞的分子和生理特性进行大规模并行分析。我们现在建议进行一项综合调查的人胎儿皮层细胞在前额叶，运动，和视觉皮层分类细胞类型和谱系。我们的工作将通过解决三个具体目标来阐明人类皮层细胞多样性的发育起源。 首先，我们将使用无偏的细胞类型分类来提供特定脑区域中定义的祖细胞和未成熟细胞类型的数量的现实估计。我们假设，祖细胞的多样性影响皮层区域的结构和连接差异的发展，但祖细胞和成年神经元的多样性之间的关系一直很难在人脑中研究。我们建议对从发育皮层的特定层收集的> 100，000个单细胞进行测序，并使用细胞条形码确定单细胞基因表达，以进行有效的低覆盖率mRNA测序。通过分析基因、microRNA和染色质状态，我们期望能够区分祖细胞和有丝分裂后细胞的离散群体。第二，我们假设，生理和分子特性的结合理解将改善细胞类型分类，并揭示功能成熟的最预测分子因子。为此，我们将使用一种新的高通量筛选来测量直接捕获在微流控芯片上的单细胞中的一系列配体和神经递质的生理反应。然后将细胞裂解，mRNA逆转录，扩增和测序。这种对单细胞进行分析和分类的方法将整合有关分子特性的信息与跨解剖位置的生理反应。最后，我们将使用细胞发育分辨率图，将细胞分类为属于特定谱系轨迹，以及离散类别。我们将使用时移显微镜和电生理学在培养的原代人类细胞和切片中验证预测的功能和分子谱系。这种方法将进一步整合来自不同皮层区域的细胞的分子和生理身份与其谱系身份，并将提供线索，以确定成熟的人类皮层中的神经元亚型和连接模式的决定因素。

 DESCRIPTION (provided by applicant): This proposal seeks to create a single cell resolution map of the developing human neocortex. We propose to determine the number of different subtypes of neural stem and progenitor cells that generate the cerebral cortex, and then follow the developmental trajectories of the newborn neurons they produce to obtain an understanding of the diversity of cortical neurons that will ultimately form the adult cortex. We plan a novel approach to this problem by integrating surveys of single cell gene expression and physiology in human cortical cells from multiple brain regions at a series of developmental stages. In collaboration with Fluidigm Corporation, we have developed innovative strategies for massively parallel profiling of molecular and physiological properties of primary human cortical cells using microfluidic technologies, cellular barcoding, and timelapse microscopy. We now propose to conduct an integrated survey of human fetal cortical cells in prefrontal, motor, and visual cortex to classify cell types and lineages. Our work will shed light on the developmental origins of cell diversity in the human cortex by addressing three specific aims. First, we will use unbiased cell type classification to provide a realistic estimate of the number of defined progenitor and immature cell types in specific brain regions. We hypothesize that progenitor diversity influences the development of structural and connectivity differences in cortical areas, but the relationship between the diversity of progenitor cells and adult neurons has been difficult to study in the human brain. We propose to sequence >100,000 single cells collected from specific lamina of the developing cortex and determine single cell gene expression using cellular barcodes for efficient low-coverage mRNA sequencing. By analyzing genes, microRNAs, and chromatin states, we anticipate to be able to distinguish discrete populations of progenitor and postmitotic cells. Second, we hypothesize that a combined understanding of physiological and molecular properties will improve cell type classification and reveal molecular factors most predictive of functional maturation. To this end we will use a novel high-throughput screen to measure physiological responses to a range of ligands and neurotransmitters in single cells captured directly on microfluidic chips. The cells will then be lysed, and mRNA reverse-transcribed, amplified, and sequenced. This approach to profiling and classification of single cells will integrate information on molecular properties with physiological responses across anatomical locations. Finally, we will classify cells as belonging to specific lineage trajectories, in additin to discrete categories, using cellular resolution maps of development. We will validate predicted functional and molecular lineages using timelapse microscopy and electrophysiology in cultured primary human cells and slices. This approach will further integrate the molecular and physiological identity of cells from distinct cortical areas with their lineage identity and will provide clues to the determinants that specify neuronal subtypes and connectivity patterns in the maturing human cortex.