title: Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants.
abstract: Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Here a new test format was established. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of the stopper barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of obtaining data on viability and migration by automated image processing was addressed by developing a freeware. Data on cell proliferation were obtained by labelling replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as an experimental confounder was tested experimentally by performing the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allows classification of toxicants as either inactive, leading to unspecific cytotoxicity, or specifically inhibiting NC migration at non-cytotoxic concentrations.
authors.affiliation: In vitro Toxicology and Biomedicine, Dept inaugurated by the Doerenkamp-Zbinden Foundation at the University of Konstanz, Konstanz, Germany.|Research Training Group RTG1331, University of Konstanz, Konstanz, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Nyffeler
authors.affiliation: In vitro Toxicology and Biomedicine, Dept inaugurated by the Doerenkamp-Zbinden Foundation at the University of Konstanz, Konstanz, Germany.
authors.forename: Christiaan
authors.initials: C
authors.lastname: Karreman
authors.affiliation: In vitro Toxicology and Biomedicine, Dept inaugurated by the Doerenkamp-Zbinden Foundation at the University of Konstanz, Konstanz, Germany.
authors.forename: Heidrun
authors.initials: H
authors.lastname: Leisner
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.|Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Yong Jun
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.|Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Gabsang
authors.initials: G
authors.lastname: Lee
authors.affiliation: In vitro Toxicology and Biomedicine, Dept inaugurated by the Doerenkamp-Zbinden Foundation at the University of Konstanz, Konstanz, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Waldmann
authors.affiliation: In vitro Toxicology and Biomedicine, Dept inaugurated by the Doerenkamp-Zbinden Foundation at the University of Konstanz, Konstanz, Germany.|Research Training Group RTG1331, University of Konstanz, Konstanz, Germany.|Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Leist
mesh_terms: D054443:Cell Migration Assays / Q000379:methods*; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation; D002470:Cell Survival; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D009432:Neural Crest / Q000166:cytology / Q000187:drug effects*; D013234:Stem Cells; D013723:Teratogens / Q000633:toxicity
chemical_list: D013723:Teratogens
keywords:  high content imaging; cell proliferation; cell tracking; developmental toxicity; human stem cells