Human neural progenitor cell differentiation in vitro

This experiment was designed to characterize the temporal gene expression dynamics of differentiating human neural progenitor cells through time in culture. In vitro models of neuronal differentiation are emerging as an important tool for high-throughput and high-content screening in neurodevelopmental toxicology. However, little has been done to characterize normal temporal pathway dynamics of differentiation in vitro or to anchor processes captured in vitro to developmental processes in vivo that are vulnerable to toxicant perturbation. We cultured human neural progenitor cell (hNPCs) up to 21 days in differentiation conditions, examining changes in morphology, protein expression and global gene expression. Over time, hNPCs acquired morphological characteristics of mature neuronal networks and increased protein expression of neuronal markers, including beta tubulin III, MAP2, and alpha-synuclein. Significantly changed genes were organized according to temporal expression patterns using K-means clustering, revealing 3 phases of gene expression. Quantitative pathway analysis identified gene ontology (GO) terms enriched among genes expressed in each of these phases and created a quantitative summary of temporal pathway trends in vitro. These observations of morphology, protein and gene expression provide a timeline of progression through differentiation, facilitating identification of key phases of sensitivity. We compared gene expression in vitro with publicly available gene expression data from developing human brain tissue in vivo and found substantial concordance in relative gene expression intensity. Genes highly expressed in both samples were enriched for key processes of brain development, including proliferation, migration, differentiation, synapse formation, and neurotransmission. GO terms enriched among genes highly expressed only in vivo or only in vitro reveal important differences between systems. For example, genes highly expressed in vitro are enriched for more stress and apoptosis pathways. This analysis provides a temporal roadmap of in vitro neuronal differentiation and anchors gene expression patterns in vitro to gene expression during sensitive windows of in vivo development. By anchoring in vitro dynamics to in vivo reference points, this work clarifies the extent to which fundamental processes of brain development are captured in our model. hNPCs were cultured in vitro in differentiation conditions. To characterize temporal gene expression dynamics in differentiating cultures, gene expression was measured 0, 1, 3, 7, 14 and 21 days following initiation of differentiation. Three replicate samples from independent experiments were collected for each timepoint.本实验旨在表征培养体系中分化的人类神经祖细胞（human neural progenitor cells, hNPCs）的时序基因表达动态。神经元分化体外模型正逐渐成为神经发育毒理学领域高通量、高内涵筛选的重要工具。然而，目前针对体外分化过程的正常时序通路动态的研究仍较为匮乏，且鲜有研究将体外捕获的生物学过程与体内易受毒物扰动的发育过程建立对应关联。 我们在分化培养条件下对hNPCs进行长达21天的培养，对其形态学特征、蛋白质表达及全局基因表达的变化进行检测。随着培养时间推移，hNPCs逐渐呈现出成熟神经元网络的形态学特征，神经元标志物（包括β微管蛋白III、MAP2以及α-突触核蛋白）的蛋白质表达水平亦显著升高。 研究采用K均值聚类对显著差异表达基因按时序表达模式进行分组，成功揭示了三类基因表达时相。通过定量通路分析，我们鉴定出各时相基因中富集的基因本体（Gene Ontology, GO）术语，并构建了体外时序通路趋势的定量总结。 上述关于形态、蛋白质及基因表达的观测结果提供了一套完整的神经元分化时序进程图谱，有助于精准识别分化过程中的关键敏感时相。我们将体外基因表达数据与公开的体内发育人脑组织基因表达数据集进行比对，发现二者在相对基因表达强度上具有高度一致性。在两种样本中均高表达的基因显著富集于脑发育的关键生物学过程，包括细胞增殖、迁移、分化、突触形成以及神经递质传递。 仅在体内或仅在体外高表达的基因所富集的GO术语，则揭示了两类实验系统间的重要差异。例如，体外高表达的基因显著富集于更多应激与凋亡相关通路。 本分析提供了体外神经元分化的完整时序路线图，并将体外基因表达模式与体内发育敏感窗口期的基因表达建立了锚定关联。通过将体外分化的动态过程与体内参考基准进行锚定，本研究阐明了本模型所覆盖的脑发育基本过程的范围与程度。 本研究中，hNPCs均在体外分化培养条件下进行培养。为表征分化培养体系的时序基因表达动态，我们在分化启动后的第0、1、3、7、14及21天共6个时间点进行了基因表达检测。每个时间点均收集来自独立重复实验的三份样本。