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

Karin Lauschke, Andreas Frederik Treschow, Mikkel Aabech Rasmussen, Nichlas Davidsen, Bjørn Holst, Jenny Emnéus, Camilla Taxvig, Anne Marie Vinggaard*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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.
Original languageEnglish
JournalArchives of Toxicology
Pages (from-to)1659-1670
Number of pages12
Publication statusPublished - 2021


  • CRISPR/Cas9
  • Human-induced pluripotent stem cell
  • Thalidomide
  • Luminescence
  • Reporter cell line assay


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