ORIGINAL RESEARCH article
Front. Toxicol.
Sec. Developmental and Reproductive Toxicology
Volume 7 - 2025 | doi: 10.3389/ftox.2025.1645842
This article is part of the Research TopicNew Approach Methodologies in Developmental and Reproductive ToxicologyView all 3 articles
Implementing a trilineage differentiation in the ReproTracker assay for improved teratogenicity assessment
Provisionally accepted
José Manuel Horcas-NietoSabine Hartvelt
Luke Flatt
Jing FangEsther LamGaonan ZhangRomy van Vliet
Marleen FeliksikTom ZwetslootConnor PhilippoGiel Hendriks
Amer Jamalpoor*- Toxys BV, Oegstgeest, Netherlands
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Exposure to teratogenic compounds during pregnancy can lead to significant birth defects. Given the considerable variation in drug responses across species, along with the financial and ethical challenges associated with animal testing, the development of advanced human-based in vitro assays is imperative for effectively identifying potential human teratogens. Previously, we developed a human induced pluripotent stem cell (hiPSCs)-based biomarker assay, ReproTracker, that follows the differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay combines morphological profiling with the assessment of time-dependent expression patterns of cell-specific biomarkers to detect developmental toxicity responses. To further increase the predictability of the assay in identifying potential teratogens, we added differentiation of hiPSCs towards neural rosette-like cells. We evaluated the performance of the extended assay with a set of 51 well-known in vivo teratogens and non-teratogens, including the compounds listed in the ICH S5 reference list. The optimized assay correctly identified (neuro)developmental toxicants that were not detected in the hepatocyte and cardiomyocyte differentiation assays. These compounds selectively downregulated gene and protein expression of the neuroectodermal marker PAX6 and/or neural rosette marker NESTIN in a concentration-dependent manner and disrupted the differentiation of hiPSCs towards neural rosette-like cells. Overall, based on the current dataset, the addition of neural commitment improved the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to 87.50%), when compared to the previously described assay. In summary, trilineage differentiation expanded the spectrum of teratogenic agents detectable by ReproTracker, making the assay an invaluable tool for early in vitro teratogenicity screening.
Keywords: developmental toxicity, New alternative method (NAM), ReproTracker, Human iPSCs, ICHS5 validation
Received: 12 Jun 2025; Accepted: 25 Aug 2025.
Copyright: © 2025 Horcas-Nieto, Hartvelt, Flatt, Fang, Lam, Zhang, van Vliet, Feliksik, Zwetsloot, Philippo, Hendriks and Jamalpoor. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Amer Jamalpoor, Toxys BV, Oegstgeest, Netherlands
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