Scientific Validation of Human Neurosphere Assays for Developmental Neurotoxicity Evaluation

Abstract

There is a call for a paradigm shift in developmental neurotoxicity (DNT) evaluation, which demands the implementation of faster, more cost-efficient, and human-relevant test systems than current in vivo guideline studies. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD), a guidance document is currently being prepared that instructs on the regulatory use of a DNT in vitro battery (DNT IVB) for fit-for-purpose applications. One crucial issue for OECD application of methods is validation, which for new approach methods (NAMs) requires novel approaches. Here, mechanistic information previously identified in vivo, as well as reported neurodevelopmental adversities in response to disturbances on the cellular and tissue level, are of central importance. In this study, we scientifically validate the Neurosphere Assay, which is based on human primary neural progenitor cells (hNPCs) and an integral part of the DNT IVB. It assesses neurodevelopmental key events (KEs) like NPC proliferation (NPC1ab), radial glia cell migration (NPC2a), neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5), and thyroid hormone-dependent oligodendrocyte maturation (NPC6). In addition, we extend our work from the hNPCs to human induced pluripotent stem cell-derived NPCs (hiNPCs) for the NPC proliferation (iNPC1ab) and radial glia assays (iNPC2a). The validation process we report for the endpoints studied with the Neurosphere Assays is based on 1) describing the relevance of the respective endpoints for brain development, 2) the confirmation of the cell type-specific morphologies observed in vitro, 3) expressions of cell type-specific markers consistent with those morphologies, 4) appropriate anticipated responses to physiological pertinent signaling stimuli and 5) alterations in specific in vitro endpoints upon challenges with confirmed DNT compounds. With these strong mechanistic underpinnings, we posit that the Neurosphere Assay as an integral part of the DNT in vitro screening battery is well poised for DNT evaluation for regulatory purposes.

Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro

“…These time points were chosen based on the endpoints of concern affected within the “Neurosphere Assay” under respective compound exposure. hNPC migration directly starts after plating the neurospheres onto the extracellular matrix and within the first 24 h hNPC migration dynamic is the highest (Baumann et al 2015 ; Koch et al 2022 ). The same refers to the time point chosen for the TM-related oligodendrocyte effect.…”

“…The same refers to the time point chosen for the TM-related oligodendrocyte effect. Sixty hours post plating, oligodendrocyte-related gene expression ( PLP1 and MBP ) increases 20–30-fold despite the low numbers of oligodendrocytes at this time point (Koch et al 2022 ). Hence, this dynamic time point is suitable for observing adverse effects on oligodendrocyte development (Klose et al 2021a ).…”

“…In the present study, we performed a case study with two selected CHMs, Lei Gong Teng (LGT; Radix Et Rhizoma Tripterygii Wilfordii , Common Threewingnut Root) and Tian Ma (TM; Gastrodia elata Blume , Tall Gastrodia Tuber) by evaluating their adverse neurodevelopmental effects with the test methods NPC1-5 (Koch et al 2022 ), which are based on developing human neural progenitor cells (hNPCs) in the “Neurosphere Assay,” which is part of a current OECD/EFSA (European Food Safety Authority) DNT in vitro battery (IVB; Masjosthusmann et al 2020 ; EFSA 2021 ). Data generated in this study are placed in an AOP context by applying one and expanding a second already existing putative AOP for DNT.…”

A human iPSC-basedin vitroneural network formation assay to investigate neurodevelopmental toxicity of pesticides

“…The copyright holder for this this version posted January 13, 2023. ; https://doi.org/10.1101/2023.01.12.523741 doi: bioRxiv preprint 3 cell (hNPC) proliferation (Baumann et al, 2014(Baumann et al, , 2015Harrill et al, 2018;Nimtz et al, 2019;Masjosthusmann et al, 2020;Koch et al, 2022) and apoptosis (Druwe et al, 2015;Harrill et al, 2018), cell migration (Baumann et al, 2015(Baumann et al, , 2016Nyffeler et al, 2017;Schmuck et al, 2017;Masjosthusmann et al, 2020;Koch et al, 2022), hNPC-neuronal (Baumann et al, 2015;Schmuck et al, 2017;Masjosthusmann et al, 2020;Koch et al, 2022) and oligodendrocyte differentiation (Fritsche et al, 2015;Dach et al, 2017;Schmuck et al, 2017;Masjosthusmann et al, 2020;Klose et al, 2021;Koch et al, 2022), neurite outgrowth (human: Harrill et al, 2010Harrill et al, , 2018Krug et al, 2013;Hoelting et al, 2016;Masjosthusmann et al, 2020;Koch et al, 2022;rat: Harrill et al, 2013rat: Harrill et al, , 2018, as well as neuronal maturation and synaptogenesis (rat: Harrill et al, 2011Harrill et al, , 2018.…”

“…Following this consensus, a DNT in vitro battery (IVB) was compiled, which includes not one, but various DNT test methods, covering different neurodevelopmental processes, so-called key events (KEs), and developmental stages to approximate the complexity of human brain development (Fritsche, 2017; Fritsche, Crofton, Hernandez, Bennekou, et al, 2017; Bal-Price et al, 2018). Within this DNT IVB, neurodevelopment is described by in vitro assays covering the following KEs: human neural progenitor cell (hNPC) proliferation (Baumann et al, 2014, 2015; Harrill et al, 2018; Nimtz et al, 2019; Masjosthusmann et al, 2020; Koch et al, 2022) and apoptosis (Druwe et al, 2015; Harrill et al, 2018), cell migration (Baumann et al, 2015, 2016; Nyffeler et al, 2017; Schmuck et al, 2017; Masjosthusmann et al, 2020; Koch et al, 2022), hNPC-neuronal (Baumann et al, 2015; Schmuck et al, 2017; Masjosthusmann et al, 2020; Koch et al, 2022) and oligodendrocyte differentiation (Fritsche et al, 2015; Dach et al, 2017; Schmuck et al, 2017; Masjosthusmann et al, 2020; Klose et al, 2021; Koch et al, 2022), neurite outgrowth ( human : Harrill et al, 2010, 2018; Krug et al, 2013; Hoelting et al, 2016; Masjosthusmann et al, 2020; Koch et al, 2022; rat : Harrill et al, 2013, 2018), as well as neuronal maturation and synaptogenesis ( rat : Harrill et al, 2011, 2018).…”