A novel human pluripotent stem cell-based assay to predict developmental toxicity

Lauschke, Karin; Rosenmai, Anna Kjerstine; Meiser, Ina; Neubauer, Julia C.; Schmidt, Katharina; Rasmussen, Mette; Holst, Bjørn; Taxvig, Camilla; Emnéus, Jenny; Vinggaard, Anne Marie

doi:10.1007/s00204-020-02856-6

Cited by 23 publications

(34 citation statements)

References 49 publications

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“…The valproic acid (P4543, Sigma-Aldrich) stock solution was prepared in ethanol at 600 mM and diluted in ethanol 1000 × of the test concentrations (25, 50, 100, 200 and 300 µM). We showed previously that both compounds are not cytotoxic at these concentrations in our cells (Lauschke et al 2020 ).…”

Section: Methodssupporting

confidence: 57%

“…Human-induced pluripotent stem cells were differentiated into cardiomyocytes as described in Lauschke et al ( 2020 ). Essentially, hiPSC were harvested as single cells by incubation in Gibco™ TrypLE™ Select (Thermo Fisher Scientific) for 1–2 min.…”

Section: Methodsmentioning

confidence: 99%

“…This process mimics the first 3 weeks of human fetal development including the first heart beats on day 21 (Spielmann et al 1997 ). Based on this principle, we previously developed the PluriBeat assay with human induced pluripotent stem cells (hiPSC) (Lauschke et al 2020 ). The PluriBeat assay employs an 8-day protocol, in which hiPSC are aggregated into EBs and differentiated into beating cardiomyocytes.…”

Section: Introductionmentioning

confidence: 99%

“…The PluriBeat assay employs an 8-day protocol, in which hiPSC are aggregated into EBs and differentiated into beating cardiomyocytes. EBs are exposed to test chemicals for the last 5 days and beating is scored and compared to control conditions (Lauschke et al 2020 ). However, scoring of beating EBs is time consuming and labor intensive, as it requires visual inspection of each EB with a light microscope.…”

Section: Introductionmentioning

confidence: 99%

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Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

et al. 2021

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To test large numbers of chemicals for developmental toxicity, rapid in vitro tests with standardized readouts for automated data acquisition are needed. However, the most widely used assay, the embryonic stem cell test, relies on the counting of beating embryoid bodies by visual inspection, which is laborious and time consuming. We previously developed the PluriBeat assay based on differentiation of human induced pluripotent stem cells (hiPSC) that we demonstrated to be predictive for known teratogens at relevant concentrations using the readout of beating cardiomyocytes. Here, we report the development of a novel assay, which we term the PluriLum assay, where we have introduced a luciferase reporter gene into the locus of NKX2.5 of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established two NKX2.5 reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineered NKX2.5 reporter cell line differentiated into cardiomyocytes with the same efficiency as the original wild-type line. We then exposed the cells to valproic acid (25–300 μM) and thalidomide (0.1–36 µM) and compared the PluriBeat readout of the cardiomyocytes with the luminescence intensity of the PluriLum assay. The results showed that thalidomide decreased luminescence intensity significantly with a higher potency and efficacy compared to the beating readout. With this, we have developed a novel hiPSC-based assay with a standardized readout that may have the potential for higher throughput screening for developmental toxicity.

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Section: Methodssupporting

confidence: 57%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

et al. 2021

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“…Cell-based tests, known as in vitro techniques, are commonly used to assess the safety and toxicity of drugs and chemicals in the replacement of animal testing [ 22 ]. In vitro studies are capable of providing faster, inexpensive, and valuable information that helps researchers to assess the potential risks or possible toxicity of newly developed nanomaterials before their final application [ 17 ].…”

Section: Category Of In Vitro Modelsmentioning

confidence: 99%

Current Strategies in Assessment of Nanotoxicity: Alternatives to In Vivo Animal Testing

Huang

Lee

Hsu

et al. 2021

IJMS

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Millions of experimental animals are widely used in the assessment of toxicological or biological effects of manufactured nanomaterials in medical technology. However, the animal consciousness has increased and become an issue for debate in recent years. Currently, the principle of the 3Rs (i.e., reduction, refinement, and replacement) is applied to ensure the more ethical application of humane animal research. In order to avoid unethical procedures, the strategy of alternatives to animal testing has been employed to overcome the drawbacks of animal experiments. This article provides current alternative strategies to replace or reduce the use of experimental animals in the assessment of nanotoxicity. The currently available alternative methods include in vitro and in silico approaches, which can be used as cost-effective approaches to meet the principle of the 3Rs. These methods are regarded as non-animal approaches and have been implemented in many countries for scientific purposes. The in vitro experiments related to nanotoxicity assays involve cell culture testing and tissue engineering, while the in silico methods refer to prediction using molecular docking, molecular dynamics simulations, and quantitative structure–activity relationship (QSAR) modeling. The commonly used novel cell-based methods and computational approaches have the potential to help minimize the use of experimental animals for nanomaterial toxicity assessments.

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A novel human stem cell‐based biomarker assay for in vitro assessment of developmental toxicity

Jamalpoor¹,

Hartvelt²,

Dimopoulou³

et al. 2022

Birth Defects Research

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Background Testing for developmental toxicity according to the current regulatory guidelines requires large numbers of animals, making these tests very resource intensive, time‐consuming, and ethically debatable. Over the past decades, several alternative in vitro assays have been developed, but these often suffered from low predictability and the inability to provide a mechanistic understanding of developmental toxicity. Methods To identify embryotoxic compounds, we developed a human induced pluripotent stem cells (hiPSCs)‐based biomarker assay. The assay is based on the differentiation of hiPSCs into functional cardiomyocytes and hepatocytes. Proper stem cell differentiation is investigated by morphological profiling and assessment of time‐dependent expression patterns of cell‐specific biomarkers. In this system, a decrease in the expression of the biomarker genes and morphology disruption of the differentiated cells following compound treatment indicated teratogenicity. Results The hiPSCs‐based biomarker assay was validated with 21 well‐established in vivo animal teratogenic and non‐teratogenic compounds during cardiomyocyte and hepatocyte differentiation. The in vivo teratogenic compounds (e.g., thalidomide and valproic acid) markedly disrupted morphology, functionality, and the expression pattern of the biomarker genes in either one or both cell types. Non‐teratogenic chemicals generally had no effect on the morphology of differentiated cells, nor on the expression of the biomarker genes. Compared to the in vivo classification, the assay achieved high accuracy (91%), sensitivity (91%), and specificity (90%). Conclusion The assay, which we named ReproTracker®, is a state‐of‐the‐art in vitro method that can identify the teratogenicity potential of new pharmaceuticals and chemicals and signify the outcome of in vivo test systems.

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A novel human pluripotent stem cell-based assay to predict developmental toxicity

Cited by 23 publications

References 49 publications

Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

Current Strategies in Assessment of Nanotoxicity: Alternatives to In Vivo Animal Testing

A novel human stem cell‐based biomarker assay for in vitro assessment of developmental toxicity

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