The DNT-EST: A predictive embryonic stem cell-based assay for developmental neurotoxicity testing in vitro

Hayeß, K.; Riebeling, Christian; Pirow, Ralph; Steinfath, Matthias; Sittner, D.; Slawik, B.; Luch, Andreas; Seiler, A.

doi:10.1016/j.tox.2013.09.012

Cited by 35 publications

(12 citation statements)

References 71 publications

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“…It would be considered as a great improvement to be able to gather experiments together to evaluate embryotoxicity. Other laboratories proposed new protocols considering the neuronal (Baek et al, 2012;Hayess et al, 2013) or the bone tissue (de Jong et al, 2012;zur Nieden et al, 2010). However, although showing promise and representing real advances in the domain, those protocols still require complicated manipulations that can hardly be automated with the current available technologies.…”

Section: Discussionmentioning

confidence: 99%

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

et al. 2015

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-The embryonic stem cell test (EST) is a promising alternative method for evaluating embryotoxicity of test chemicals by measuring cytotoxicity and differentiation toxicity using mouse ES cells. Differentiation toxicity is analyzed by microscopically counting the beating of embryonic bodies after 10 days of culture. However, improvements are necessary to reduce the laborious manipulations involved and the time required to obtain results. We have previously reported the successful stable transfection of ES cells (ES-D3) with the heart and neural crest derivatives expressed transcript 1 (Hand1) gene and the establishment of a 96-well multi-plate-based new EST with luciferase reporter assay 6 days after treatment with test chemicals. Now, we propose an even more rapid and easier EST, named Hand1-Luc EST. We established another cell line to monitor the Hand1 gene expression via a luciferase reporter gene. By mRNA analysis and luciferase assay, we examined in detail the luciferase activity during cell differentiation, which allowed us to reduce the time of measurement from day 6 to day 5 (120 hr). Furthermore, the protocol was improved, with, among others, the measurement of cytotoxicity and differentiation toxicity taking place in the same 96-well round bottom plate instead of two different plates. With the positive control, 5-fluorouracil (5-FU), and 9 test chemicals, data with high reproducibility and very low variation (CV < 50%) in the relevant endpoints were obtained. This study shows that the Hand1-Luc EST could provide an accurate and sensitive short-term test for prediction of embryotoxicants by measuring cytotoxicity and differentiation toxicity from the same sample.

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Section: Discussionmentioning

confidence: 99%

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

et al. 2015

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“…The potential of using murine embryonic stem cells (mESCs) for in vitro developmental toxicity testing was recognized long time ago (Heuer et al 1993), and the murine ESC-based embryonic stem cell test (EST) became a validated platform for embryotoxicology testing (Genschow et al 2004;Hayess et al 2013). Robust protocols have been developed for neural differentiation of mESC and are in use for DNT (Kuegler et al 2010;Zimmer et al 2011a, b;Theunissen et al 2012;Visan et al 2012;Smirnova et al 2014a, c;Colaianna et al 2016).…”

Section: Non-human Primary Neural Cells Neural Cell Lines and Embrymentioning

confidence: 99%

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

et al. 2016

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Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

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“…These include neurite outgrowth assays using human neuronal cell models that demonstrated selective effects of chemicals on neurite outgrowth in the absence of cytotoxicity 24 and species differences in chemical sensitivity, 20 assays for chemical effects on network function, 25 synaptogenesis, 26 cell migration, 27 and cell differentiation. 28,29 However, these assays have not yet been utilized to test ToxCast chemicals or other similar large chemical libraries. Improving the ability to predict neurotoxicity and/or developmental neurotoxicity of environmental chemicals will depend on further consideration of the present results in the context of other ToxCast assays, screening of ToxCast compounds using additional assays for neural-related endpoints, and evaluation of the results in the context of vivo databases (e.g., ToxRefDb).…”

Section: Discussionmentioning

confidence: 99%

Use of Neural Models of Proliferation and Neurite Outgrowth to Screen Environmental Chemicals in the ToxCast Phase I Library

RadioNicholas

BreierJoseph

ReifDavid

et al. 2015

Applied In Vitro Toxicology

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The U.S. Environmental Protection Agency's ToxCast program aims to develop rapid and cost-effective toxicity testing approaches. As part of this effort, we screened the ToxCast Phase I library of 309 chemicals using previously developed and optimized high-content imaging and analysis assays for proliferation in human ReNcell CX neuroprogenitor cells and neurite outgrowth in PC12 cells. Cytotoxicity was determined concurrently in both models. For the initial screening, cells were exposed in triplicate to chemicals at 40 lM for 24 (proliferation) or 96 hr (neurite outgrowth). Chemicals were deemed active if effects on any endpoint were > 3 · the standard deviation of control means. A total of 130/309 chemicals (42.1%) altered at least one of the four endpoints examined. In ReNcell CX cells, 126 (41%) of chemicals were active, and 46 (15%) were active in PC12 cells. In ReNcell CX cells, 63 chemicals selectively inhibited proliferation. In PC12 cells, only four chemicals (methyl isothiocyanate, phosalone, prodiamine, and pyridaben) selectively increased neurite outgrowth, while none selectively decreased neurite outgrowth. All active chemicals were re-tested in a concentration-response study (1 nM-40 lM) and similar results were obtained. These results demonstrate (1) high reproducibility and concordance for both assays; (2) that the sensitivity of single-concentration screening may be limited by biological variability; (3) that, for environmental compounds, cytotoxicity must be used in the interpretation of effects on selected cellular endpoints; and (4) that proliferation in human neuroprogenitor cells responds similarly to proliferation in nonneuronal human cells, but has higher specificity when concurrent cytotoxicity data are considered.

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The DNT-EST: A predictive embryonic stem cell-based assay for developmental neurotoxicity testing in vitro

Cited by 35 publications

References 71 publications

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

Use of Neural Models of Proliferation and Neurite Outgrowth to Screen Environmental Chemicals in the ToxCast Phase I Library

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