Using single cell RNAseq to unveil characteristics of CD133+ neural stem cells during development

使用单细胞 RNAseq 揭示 CD133 神经干细胞发育过程中的特征

基本信息

批准号：
9751415
负责人：
Weihong Ge
金额：
$ 19.5万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9751415
关键词：
Adult Anatomy Antibodies Atlases Biological Assay Biotechnology Brain Cell Differentiation process Cell Lineage Cell surface Cells Characteristics Complex Data Data Analyses Databases Development Embryo Emotions Endothelium Ependymal Cell Forebrain Development Future Gene Expression Regulation Genes Genetic Transcription Genomics Glial Fibrillary Acidic Protein Goals Grant Hematopoietic Heterogeneity Immunohistochemistry In Situ Hybridization Individual Knowledge Label Light Location Maps Mesenchymal Messenger RNA Microspheres Mitotic Molecular Molecular Profiling Movement Mus Nervous system structure Neural Tube Closure Neuraxis Neuroepithelial Neurons Pathway Analysis Population Property Prosencephalon Proteins Publishing Regulator Genes Reporter Reporting Resolution Stem cells Structure Surface Synapses Techniques Technology Time Tissues Tomatoes Tube Validation Vascular Endothelial Growth Factors Ventricular Work base brain cell cell cortex cell type clinical application cost experimental study genetic signature injured loss of function multipotent cell nerve stem cell nervous system development nestin protein neural circuit neurodevelopment neurogenesis novel progenitor programs prominin ranpirnase relating to nervous system repaired self-renewal single cell sequencing single-cell RNA sequencing stem cell niche stem cell population transcriptome transcriptome sequencing

项目摘要

Using single cell RNAseq to unveil characteristics of CD133+ neural stem cells during development Project Summary Neural stem cells (NSCs), by definition, are self-renewing, multipotent cells that generate major cell types of the nervous system. Our current knowledge about NSC is still quite limited, insufficient to fulfill the goal of achieving good understanding of neurodevelopment or to engage endogenous NSCs to repair degenerated or injured central nervous system (CNS). Like other tissue specific stem cells, NSCs reside in highly complex cellular microenvironment and are in close contact with both stem cell niche and their downstream progeny cell types, which had made the characterization of the cellular and molecular signatures of NSCs extremely challenging. Recently developed single cell RNAseq technology pave the way to uncover molecular characteristics of any cell types including stem cells with precision. Particularly, the novel droplet-based technique allowed thousands of single cells to be sequenced in one tube yet still produce transcriptomes with single cell resolution. In this application, we propose to apply this scalable and low cost single cell RNAseq analysis developed by 10X Genomics to characterize CD133 positive ependymal cells lining the ventricular surface of the brain. The transciptome data of thousands of single CD133+ cells from cortex at different time points during development will be subjected to comprehensive data analyses, including Weighted Gene Coexpression Network Analysis (WGCNA), Pseudo-time analysis, principle component analysis (PCA) / t- Distributed Stochastic Neighbor Embedding (tSNE). We aim to characterize the molecular features unique to CD133+ cell subtypes in a temporally and spatially specific manner, through which we can obtain a better understanding of NSCs activities during development, as well as their potential interactions with the vasculature niche. Immunohistochemistry and/or in situ hybridization analyses will be performed to validate the sequencing result and provide physical locations of each of the specific cell types. Through lineage tracing studies, we will be able to map out lineage relationships of heterogeneous NSC populations. We believe this study will provide valuable information for building the atlas or roadmap of the origins of NSCs and how they evolve during development. Moreover, we aim to bridge this project into a RO1 grant where the function of some of the interesting core regulatory genes identified by this study will be further studied by gain- and loss-of function analyses and lineage tracing studies.

使用单细胞 RNAseq 揭示 CD133+ 神经干细胞发育过程中的特征项目概要根据定义，神经干细胞 (NSC) 是一种自我更新的多能细胞，可产生以下主要细胞类型神经系统。我们目前对 NSC 的了解还相当有限，不足以实现 NSC 的目标获得对神经发育的良好理解或利用内源性 NSC 来修复退化或中枢神经系统（CNS）受伤。与其他组织特异性干细胞一样，NSC 存在于高度复杂的细胞中。细胞微环境，与干细胞生态位及其下游子代细胞密切接触类型，这使得 NSC 的细胞和分子特征的表征变得极其重要具有挑战性的。最近开发的单细胞 RNAseq 技术为揭示分子生物学铺平了道路任何细胞类型（包括干细胞）的精确特征。特别是，新型的基于液滴的该技术允许在一个试管中对数千个单细胞进行测序，但仍然产生转录组单细胞分辨率。在此应用中，我们建议应用这种可扩展且低成本的单细胞 RNAseq 10X Genomics 开发的分析用于表征心室衬里的 CD133 阳性室管膜细胞大脑表面。不同时间皮层数千个单个CD133+细胞的转录组数据开发过程中的点将接受全面的数据分析，包括加权基因共表达网络分析（WGCNA）、伪时间分析、主成分分析（PCA）/t- 分布式随机邻域嵌入 (tSNE)。我们的目标是表征独特的分子特征以时间和空间特异性的方式识别CD133+细胞亚型，通过这种方式我们可以获得更好的了解 NSC 在发展过程中的活动，以及它们与脉管系统生态位。将进行免疫组织化学和/或原位杂交分析来验证测序结果并提供每种特定细胞类型的物理位置。通过血统追溯通过研究，我们将能够绘制异质 NSC 群体的谱系关系。我们相信这一点研究将为构建 NSC 起源及其如何形成图集或路线图提供有价值的信息。在发展过程中不断发展。此外，我们的目标是将这个项目纳入 RO1 赠款，其中本研究确定的一些有趣的核心调控基因将通过增益和丢失进一步研究功能分析和谱系追踪研究。