The Human Induced Pluripotent Stem Cell Test as an Alternative Method for Embryotoxicity Testing

Abstract

The evaluation of substances for their potency to induce embryotoxicity is controlled by safety regulations. Test guidelines for reproductive and developmental toxicity rely mainly on animal studies, which make up the majority of animal usage in regulatory toxicology. Therefore, there is an urgent need for alternative in vitro methods to follow the 3R principles. To improve human safety, cell models based on human cells are of great interest to overcome species differences. Here, human induced pluripotent stem cells (hiPSCs) are an ideal cell source as they largely recapitulate embryonic stem cells without bearing ethical concerns and they are able to differentiate into most cell types of the human body. Here, we set up and characterized a fetal bovine serum (FBS)-free hiPSC-based in vitro test method, called the human induced pluripotent stem cell test (hiPS Test), to evaluate the embryotoxic potential of substances. After 10 days in culture, hiPSCs develop into beating cardiomyocytes. As terminal endpoint evaluations, cell viability, qPCR analyses as well as beating frequency and area of beating cardiomyocytes by video analyses are measured. The embryotoxic positive and non-embryotoxic negative controls, 5-Fluorouracil (5-FU) and Penicillin G (PenG), respectively, were correctly assessed in the hiPS Test. More compounds need to be screened in the future for defining the assay’s applicability domain, which will inform us of the suitability of the hiPS Test for detecting adverse effects of substances on embryonic development.

Investigating the applicability domain of the hiPSC-based PluriLum assay: an embryotoxicity assessment of chemicals and drugs

“…5-FU is the best described compound in comparable assays. In a 3D hiPSC assay, an effect at 4.5 µM was reported (Aikawa 2020 ), while 2D systems reported an effect at 3 µM (Jamalpoor et al 2022 ), at 1 µM (Galanjuk et al 2022 ), and at 0.018 nM (Walker et al 2021 ). When measuring reporter gene expression, slightly higher sensitivity is generally observed.…”

The Current Status and Work of Three Rs Centres and Platforms in Europe*

“…Currently, methods for evaluating embryotoxicity (hiPS test), 47 which is based on the ability of human induced pluripotent stem cells (hiPSCs) to differentiate into beating cardiomyocytes and test methods for evaluating adult neurotoxicity by using neurally-induced hiPSC, are being developed within the CERST-NRW project funded by the Ministry for Culture and Science of the State of North-Rhine Westphalia, Germany. CERST-NRW also funds the set-up of a biostatistical evaluation pipeline for the transparent analysis of screening data in collaboration with the Brunel University, London.…”

The Mechanism of Houttuynia cordata Embryotoxicity Was Explored in Combination with an Experimental Model and Network Pharmacology

“…With the increase in adverse events of medicinal and edible plants, the evaluation of the toxicity and safety of medicinal and edible plants has increased. Among liver, kidney, heart, and other toxicities, embryotoxicity is the most important, because it may endanger the health and life of offspring in addition to its adverse effects, and once detected [ 4 ], it is impossible to recover after discontinuation of the drug.…”