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.