Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing
Autor: | Bjørn Holst, Mikkel A. Rasmussen, Andreas Frederik Treschow, Karin Lauschke, Camilla Taxvig, Anne Marie Vinggaard, Nichlas Davidsen, Jenny Emnéus |
---|---|
Jazyk: | angličtina |
Rok vydání: | 2021 |
Předmět: |
0301 basic medicine
Luminescence Human- induced pluripotent stem cell Health Toxicology and Mutagenesis Induced Pluripotent Stem Cells Developmental toxicity Embryoid body Biology Toxicology Cell Line 03 medical and health sciences 0302 clinical medicine Genes Reporter Toxicity Tests Human-induced pluripotent stem cell Humans CRISPR Myocytes Cardiac Induced pluripotent stem cell CRISPR/Cas9 Cells Cultured Embryonic Stem Cells Reporter gene Reporter cell line assay Cell Differentiation General Medicine Embryonic stem cell In vitro Cell biology Thalidomide In Vitro Systems Teratogens 030104 developmental biology Cell culture 030217 neurology & neurosurgery |
Zdroj: | Lauschke, K, Treschow, A F, Rasmussen, M A, Davidsen, N, Holst, B, Emnéus, J, Taxvig, C & Vinggaard, A M 2021, ' Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing ', Archives of Toxicology, vol. 95, pp. 1659-1670 . https://doi.org/10.1007/s00204-021-03018-y Archives of Toxicology |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |