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

使用单细胞RNAseq揭示CD 133+神经干细胞在发育过程中的特征 项目摘要 根据定义，神经干细胞（NSC）是自我更新的多能细胞，其产生主要的细胞类型， 神经系统我们目前对国家安全委员会的认识还很有限，不足以实现 实现对神经发育的良好理解或使内源性NSC参与修复退化或 中枢神经系统（CNS）损伤。与其他组织特异性干细胞一样，NSC存在于高度复杂的组织中。 细胞微环境，并与干细胞龛及其下游后代细胞密切接触 类型，这使得神经干细胞的细胞和分子特征的表征非常 挑战性最近开发的单细胞RNAseq技术为揭示分子生物学的分子机制铺平了道路。 包括干细胞在内的任何细胞类型的特征。特别是，新颖的基于液滴的 这项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。在该应用中，我们提出应用这种可扩展且低成本的单细胞RNAseq 由10 X Genomics开发的分析，用于表征心室内衬的CD 133阳性室管膜细胞 大脑的表面。不同时间数千个单个皮质CD 133+细胞的转录组数据 开发过程中的点将进行全面的数据分析，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- 分布式随机邻居嵌入（tSNE）。我们的目标是表征独特的分子特征， CD 133+细胞亚型在时间和空间上的特异性，通过它我们可以获得更好的 了解神经干细胞在发育过程中的活动，以及它们与 脉管生态位将进行免疫组织化学和/或原位杂交分析，以验证 测序结果并提供每种特定细胞类型的物理位置。通过血统追踪 通过这些研究，我们将能够绘制出异质性NSC群体的谱系关系。我们相信这 这项研究将为建立神经干细胞起源的地图集或路线图以及它们如何 在发展过程中演变。此外，我们的目标是将这个项目连接成一个RO 1赠款， 本研究确定的一些有趣的核心调控基因将通过获得和丢失基因进行进一步研究。 功能分析和谱系追踪研究。

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.