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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\u00a0induced 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 <jats:italic>NKX2.5<\/jats:italic> of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established two <jats:italic>NKX2.5<\/jats:italic> reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineered <jats:italic>NKX2.5<\/jats:italic> 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\u2013300\u00a0\u03bcM) and thalidomide (0.1\u201336\u00a0\u00b5M) 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.<\/jats:p>","DOI":"10.1007\/s00204-021-03018-y","type":"journal-article","created":{"date-parts":[[2021,3,4]],"date-time":"2021-03-04T03:02:41Z","timestamp":1614826961000},"page":"1659-1670","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing"],"prefix":"10.1007","volume":"95","author":[{"given":"Karin","family":"Lauschke","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreas Frederik","family":"Treschow","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mikkel Aabech","family":"Rasmussen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nichlas","family":"Davidsen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bj\u00f8rn","family":"Holst","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jenny","family":"Emn\u00e9us","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Camilla","family":"Taxvig","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6822-3572","authenticated-orcid":false,"given":"Anne Marie","family":"Vinggaard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,3,4]]},"reference":[{"key":"3018_CR1","doi-asserted-by":"publisher","first-page":"201","DOI":"10.1254\/jphs.13162FP","volume":"124","author":"N Aikawa","year":"2014","unstructured":"Aikawa N, Kunisato A, Nagao K et al (2014) Detection of thalidomide embryotoxicity by in vitro embryotoxicity testing based on human iPS cells. 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