Human Neurospheres as Three-Dimensional Cellular Systems for Developmental Neurotoxicity Testing

Abstract

BackgroundDevelopmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the “3 Rs” (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening.ObjectivesThe goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells.MethodsWe established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells.ResultsWe found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress.ConclusionsHuman neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology

“…this may allow generation of physiologically relevant cellular systems in drug discovery and toxicology screenings. In addition, since neurospheres provide a microenvironment for key events during neurodevelopment such as NSC maintenance, proliferation and differentiation, the neurosphere system enables the assessment of neurodevelopmental toxicants (Fritsche et al, 2011;Moors et al, 2009).…”

“…they may then, e.g., be used for electrophysiological studies or to follow differentiation during radial migration on a laminin-coated surface (Moors et al, 2007(Moors et al, , 2009van Vliet et al, 2007). It is noteworthy that despite differentiation onto a surface, this manner of differentiation differs from simple 2D single cell plating because there is glial cell guidance with neuronal growth on top in a processorganized manner out of an organoid cell system (Ahlenius and Kokaia, 2010;Bal-Price et al, 2012;liu and Sun, 2011;Schreiber et al, 2010).…”

“…It is noteworthy that despite differentiation onto a surface, this manner of differentiation differs from simple 2D single cell plating because there is glial cell guidance with neuronal growth on top in a processorganized manner out of an organoid cell system (Ahlenius and Kokaia, 2010;Bal-Price et al, 2012;liu and Sun, 2011;Schreiber et al, 2010). this phenomenon can be seen in the organized process of cell migration, which can also be quantified in such a system (Moors et al, 2007(Moors et al, , 2009.…”

Current standing and future prospects for the technologies proposed to transform toxicity testing in the 21st century

“…Current in vivo-based test guidelines for developmental neurotoxicity are too timely and costly to test large numbers of chemicals. To develop a more efficient paradigm, embryonic and adult stem cells are extensively used to evaluate the adverse effects of chemicals on processes of neurodevelopment (Kuegler et al, 2010;Moors et al, 2009;Buzanska et al, 2009). A recent high content screening study measuring viability, proliferation, migration, and differentiation of neural progenitor cells showed promising results in prediction of in vivo developmental neurotoxicity (Breier et al, 2010).…”

Literature review on in vitro and alternative Developmental Neurotoxicity (DNT) testing methods

“…For one, primary human neural progenitor cells (hNPCs) are available from Lonza (Verviers, Belgium) and have been used to set up a testing strategy covering multiple neurodevelopmental endpoints (Moors et al, 2009;record nr. 2682record nr.…”