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Updated: Sep 8, 2025
Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing
Published on: June 9, 2017
Neuronal differentiation pathways and compound-induced developmental neurotoxicity in the human neural progenitor
Victoria C de Leeuw1, Conny T M van Oostrom2, Paul F K Wackers2
1Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands; Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, the Netherlands.
Abstract:
There is an increased awareness that the use of animals for compound-induced developmental neurotoxicity (DNT) testing has limitations. Animal-free innovations, especially the ones based on human stem cell-based models are pivotal in studying DNT since they can mimic processes relevant to human brain development. Here we present the human neural progenitor test (hNPT), a 10-day protocol in which neural progenitor cells differentiate into a neuron-astrocyte co-culture. The study aimed to characterise differentiation over time and to find neurodevelopmental processes sensitive to compound exposure using transcriptomics. 3992 genes regulated in unexposed control cultures (p ≤ 0.001, log2FC ≥ 1) showed Gene Ontology (GO-) term enrichment for neuronal and glial differentiation, neurite extension, synaptogenesis, and synaptic transmission. Exposure to known or suspected DNT compounds (acrylamide, chlorpyrifos, fluoxetine, methyl mercury, or valproic acid) at concentrations resulting in 95% cell viability each regulated unique combinations of GO-terms relating to neural progenitor proliferation, neuronal and glial differentiation, axon development, synaptogenesis, synaptic transmission, and apoptosis. Investigation of the GO-terms 'neuron apoptotic process' and 'axon development' revealed common genes that were responsive across compounds, and might be used as biomarkers for DNT. The GO-term 'synaptic signalling', on the contrary, whilst also responsive to all compounds tested, showed little overlap in gene expression regulation patterns between the conditions. This GO-term may articulate compound-specific effects that may be relevant for revealing differences in mechanism of toxicity. Given its focus on neural progenitor cell to mature multilineage neuronal cell maturation and its detailed molecular readout based on gene expression analysis, hNPT might have added value as a tool for neurodevelopmental toxicity testing in vitro. Further assessment of DNT-specific biomarkers that represent these processes needs further studies.
Insights
Animal-free developmental neurotoxicity (DNT) testing using human stem cells offers a promising alternative. The human neural progenitor test (hNPT) identifies potential biomarkers for DNT by analyzing gene expression during neural development.
Area of Science:
- Toxicology
- Neuroscience
- Stem Cell Biology
Background:
- Traditional animal testing for developmental neurotoxicity (DNT) has limitations.
- Human stem cell-based models offer a viable alternative for studying human brain development.
- The human neural progenitor test (hNPT) is a novel in vitro model.
Purpose of the Study:
- To characterize differentiation in the hNPT model over 10 days.
- To identify neurodevelopmental processes sensitive to chemical compound exposure.
- To explore potential biomarkers for DNT using transcriptomics.
Main Methods:
- Neural progenitor cells were differentiated into neuron-astrocyte co-cultures over 10 days.
- Transcriptomic analysis was performed on control and compound-exposed cultures.
- Gene Ontology (GO) term enrichment analysis identified affected biological processes.
Main Results:
- Control cultures showed GO-term enrichment for neuronal and glial differentiation, neurite extension, and synaptogenesis.
- Exposure to known DNT compounds regulated unique GO-terms related to neural development and apoptosis.
- Commonly regulated genes in 'neuron apoptotic process' and 'axon development' may serve as DNT biomarkers.
- The 'synaptic signalling' GO-term revealed compound-specific effects.
Conclusions:
- The hNPT model effectively mimics human neural development and is sensitive to DNT compound exposure.
- Specific GO-terms and genes show potential as biomarkers for DNT.
- The hNPT provides a valuable in vitro tool for DNT assessment, with further biomarker validation needed.
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