Classification of Developmental Toxicants in a Human iPSC Transcriptomics-Based Test

Abstract

Despite the progress made in developmental toxicology, there is a great need for in vitro tests that identify developmental toxicants in relation to human oral doses and blood concentrations. In the present study, we established the hiPSC-based UKK2 in vitro test and analyzed genome-wide expression profiles of 23 known teratogens and 16 non-teratogens. Compounds were analyzed at the maximal plasma concentration ( C max ) and at 20-fold C max for a 24 h incubation period in three independent experiments. Based on the 1000 probe sets with the highest variance and including information on cytotoxicity, penalized logistic regression with leave-one-out cross-validation was used to classify the compounds as test-positive or test-negative, reaching an area under the curve (AUC), accuracy, sensitivity, and specificity of 0.96, 0.92, 0.96, and 0.88, respectively. Omitting the cytotoxicity information reduced the test performance to an AUC of 0.94, an accuracy of 0.79, and a sensitivity of 0.74. A second method, which used the number of significantly deregulated probe sets to classify the compounds, resulted in a specificity of 1; however, the AUC (0.90), accuracy (0.90), and sensitivity (0.83) were inferior compared to those of the logistic regression-based procedure. Finally, no increased performance was achieved when the high test concentrations (20-fold C max ) were used, in comparison to testing within the realistic clinical range (1-fold C max ). In conclusion, although further optimization is required, for example, by including additional readouts and cell systems that model different developmental processes, the UKK2-test in its present form can support the early discovery-phase detection of human developmental toxicants.

High Accuracy Classification of Developmental Toxicants by In Vitro Tests of Human Neuroepithelial and Cardiomyoblast Differentiation

“…In addition, 5,5-diphenylhydantoin sodium salt (sc-214337, CAS# 690-93-3) and diphenhydramine hydrochloride (sc-204729, CAS# 147-24-0) were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). All compounds were dissolved and stored at 20,000-fold C max concentrations in 100% DMSO (Carl Roth, Germany) or, alternatively, in distilled water, if soluble, as in [ 14 ].…”

“…The tested concentrations (1- and 20-fold C max ) of the teratogens and non-teratogens ( Table 1 ), as well as the information on teratogenicity, correspond to a previously published study [ 14 ] with one exception: retinol (vitamin A) was included as a non-teratogen at a C max of 1 µM and as a teratogen for a C max of 20 µM. The rationale is given in the Supplemental Information .…”

“…In our most recent work [ 14 ], we used a previously-described hiPSC-based test (UKK2) that recapitulates cardiomyogenic differentiation [ 15 , 16 ]. Our procedure, based on substance-induced cytotoxicity and gene expression changes after exposure to 23 known teratogens and 16 non-teratogens at the human peak plasma concentration (C max ), resulted in an AUC, accuracy, sensitivity, and specificity of 0.96, 0.92, 0.96, and 0.88, respectively, to correctly classify compounds as teratogenic or non-teratogenic [ 14 ]. These favorable performance metrics were unexpected, since most of the tested teratogens were not previously reported to specifically compromise cardiomyogenic differentiation.…”

“…A possible explanation is that the gene regulatory networks involved in the differentiation of hiPSC to cardiomyocytes in the UKK2 protocol overlap with networks of limb and spine development. Despite the overall favorable results, the hiPSC-cardiomyocyte differentiation test (UKK2) also has some limitations; for example, the teratogen atorvastatin delivered false negative results [ 14 ]. A further limitation of the UKK2 protocol is the requirement of a relatively high concentration of the glycogen synthase kinase 3β inhibitor CHIR99021, which induces cell stress and expression changes itself.…”

“…In the present work, the same set of compounds that was previously investigated in the cardiomyocyte differentiation test UKK2 [ 14 ] was analyzed with the UKN1 test, and identical methods of gene expression profiling and statistical evaluation were applied. The following questions were addressed: (1) Can classification performance be further improved by using the UKN1 test system compared to the previously published UKK2?…”