A novel screening test to predict the developmental toxicity of drugs using human induced pluripotent stem cells

Abstract

In vitro human induced pluripotent stem (iPS) cells testing (iPST) to assess developmental toxicity, e.g., the induction of malformation or dysfunction, was developed by modifying a mouse embryonic stem cell test (EST), a promising animal-free approach. The iPST evaluates the potential risks and types of drugs-induced developmental toxicity in humans by assessing three endpoints: the inhibitory effects of the drug on the cardiac differentiation of iPS cells and on the proliferation/survival of iPS cells and human fibroblasts. In the present study, the potential developmental toxicity of drugs was divided into three classes (1: non-developmentally toxic, 2: weakly developmentally toxic and 3: strongly developmentally toxic) according to the EST criteria. In addition, the type of developmental toxicity of drugs was grouped into three types (1: non-effective, 2: embryotoxic [inducing growth retardation/dysfunction]/ deadly or 3: teratogenic [inducing malformation]/deadly) by comparing the three endpoints. The present study was intended to validate the clinical predictability of the iPST. The traditionally developmentally toxic drugs of aminopterin, methotrexate, all-trans-retinoic acid, thalidomide, tetracycline, lithium, phenytoin, 5-fluorouracil, warfarin and valproate were designated as class 2 or 3 according to the EST criteria, and their developmental toxicity was type 3. The non-developmentally toxic drugs of ascorbic acid, saccharin, isoniazid and penicillin G were designated as class 1, and ascorbic acid, saccharin and isoniazid were grouped as type 1 while penicillin G was type 2 but not teratogenic. These results suggest that the iPST is useful for predicting the human developmental toxicity of drug candidates in a preclinical setting.

A novel human pluripotent stem cell-based assay to predict developmental toxicity

“…However, in spite of that these therapeutic plasma concentrations were at a level where we detected effects on the beat score in vitro. Effective concentrations of thalidomide in in vitro assays for developmental toxicity have been reported at concentrations ranging between 0.5 and 450 µM (Mayshar et al 2011;Palmer et al 2013;Aikawa et al 2014;Kameoka et al 2014;Aikawa 2020). The response to thalidomide in the PluriBeat assay using BIONi010-C (IC 25 2.0 µM) and IMR90-1 (IC 25 14.3 µM) was comparable to previously published data.…”

“…Therefore, we decided to use hiPSC in the PluriBeat assay, since they possess no ethical concerns and can mimic the developing embryo to the same degree as ESCs. hiPSC has been shown to predict the embryotoxicity of thalidomide using the original EST protocol, but no data revealing the quality of the hiPSC culture and cardiac differentiation or the reproducibility between experiments were shown (Aikawa et al 2014;Aikawa 2020). Therefore, we cannot compare our assay to this study in terms of quality, but we clearly detected thalidomide toxicity at lower concentrations (IC 25 2.0 µM compared to IC 50 450 µM in Aikawa et al 2014 and IC 50 5.5 µM in Aikawa 2020).…”

“…Thirty-two EBs per condition, mean, and SD for three biological replicates of each cell line (BIONi010-C valproic acid n = 2). Stars indicate significant differences of respective concentrations vs. control calculated with an ordinary one-way ANOVA on means of 32 EBs per condition (868-1000 µM) (Aikawa et al 2014;Shinde et al 2016), but also below at concentrations ranging from 3 to 205 µM (Uibel et al 2010;Palmer et al 2013;Kameoka et al 2014;Aikawa 2020). The two publications by Aikawa use a similar approach as the PluriBeat assay, but because no data on the quality or reproducibility of the experiments is shown, we cannot determine the reason for this discrepancy.…”

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

“…For functional studies, so far microelectrode arrays (MEA) [ 62 , 70 , 71 ] and visual inspections of beating cardiomyocytes [ 65 , 72 , 73 , 74 ] have been performed. Due to only residual attachment of the cells in our 2D protocol, such MEA experiments are inapplicable to this test system as the cells would not reach the electrodes of an MEA chip.…”

“…An assay substituting mESCs with hiPSCs, called the iPST, resulted in a higher IC 50 value of 13 µM ( Table 1 ). Here, the ID 50 value of 4.2 µM is three times lower than the IC 50 [ 72 ]. A 2D assay using hiPSCs called the hiPSC-based EST showed an IC 50 value of 0.4 nM and an ID 50 value of 0.018 nM ( Table 1 ) which differ in one order of magnitude.…”