Analyzing human neural stem cell ontogeny by consecutive isolation of Notch active neural progenitors. Homo sapiens

Decoding heterogeneity of pluripotent stem cell (PSC)-derived neural progeny is fundamental for revealing the origin of diverse progenitors, for defining their lineages, and for identifying fate determinants driving transition through distinct potencies. Here we prospectively isolated consecutively appearing PSC-derived primary progenitors based on their Notch activation state. We first isolate early neuroepithelial cells and show their broad Notch-dependent developmental and proliferative potential. Neuroepithelial cells further yield successive Notch-dependent functional primary progenitors, from early and mid neurogenic radial glia and their derived basal progenitors, to gliogenic radial glia and adult-like neural progenitors, together recapitulating hallmarks of neural stem cell (NSC) ontogeny. Gene expression profiling reveals dynamic stage specific transcriptional patterns that may link development of distinct progenitor identities through Notch activation. Our observations provide a platform for characterization and manipulation of distinct progenitor cell types amenable for developing streamlined neural lineage specification paradigms for modeling development in health and disease. Overall design: Human embryonic stem cells (hESCs) H9 were differentiated into 5 distinct populations of neural precursor cells (NPCs) over a time course of 200 days. Each neural precursor populations was then sorted for HES5 expression based on a GFP-HES5 reporter. Both the HES5 positive and HES5 negative populations were then subjected to microarray profiling in singlicate, as well as the hESCs using GeneChipPrimeView Human Gene Expression Array

解析多能干细胞（pluripotent stem cell, PSC）来源的神经子代的异质性，对于揭示各类祖细胞的起源、明确其细胞谱系，以及鉴定驱动细胞在不同潜能状态间转变的命运决定因子，均具有基础性的重要意义。本研究基于Notch激活状态，前瞻性地分离了连续出现的PSC来源原代祖细胞。本研究首先分离了早期神经上皮细胞，并证实其具备广泛的Notch依赖性发育与增殖潜能。神经上皮细胞可进一步生成一系列连续的、依赖Notch信号的功能性原代祖细胞，涵盖早期与中期神经发生型放射状胶质细胞及其衍生的基底祖细胞，直至胶质生成型放射状胶质细胞与成体样神经祖细胞，完整复现了神经干细胞（neural stem cell, NSC）发生的标志性特征。基因表达谱分析揭示了动态的阶段特异性转录模式，这类模式或可通过Notch激活，将不同祖细胞身份的发育进程相互关联。本研究的发现为表征与操控不同祖细胞类型提供了研究平台，可用于构建标准化的神经谱系特化范式，以模拟健康与疾病状态下的发育过程。
实验整体设计：将人类胚胎干细胞（human embryonic stem cell, hESCs）H9在200天的时间进程中分化为5个不同的神经前体细胞（neural precursor cell, NPCs）群体。基于GFP-HES5报告基因，对每个神经前体细胞群体进行HES5表达水平的分选。随后以单样本分别对HES5阳性与HES5阴性群体进行芯片表达谱分析，同时使用GeneChipPrimeView人类基因表达芯片对hESCs进行检测。