High accuracy classification of developmental toxicants by in vitro tests of human neuroepithelial and cardiomyoblast differentiation

Stem cell-based tests for developmental toxicity are currently intensively studied. Here, we used a protocol that differentiates human induced pluripotent stem cells (hiPSCs) to neural ectodermal progenitor cells (UKN1) and exposed them to 23 teratogens and 16 non-teratogens at human relevant concentrations. Substance-induced, genome-wide expression profiles were analyzed by two procedures that classified the compounds either based on the number of significantly deregulated genes or on a classification technique with cross-validation. Both procedures demonstrated a high performance with accuracy, sensitivity and specificity of 0.90, 0.83 and 1.0, as well as 0.87, 0.92 and 0.80, respectively. Comparing the present findings with the UKN1 test to those from our recently published UKK2 test that instead recapitulates cardiomyogenic differentiation showed a high congruence in compound classification. This may be explained by the fact that the same pathways, such as PI3K-Akt, P53, TGF-beta, MAPK, EGFR and Hippo were overrepresented among the differentially expressed genes for both tests. However, for some teratogens discrepant results were obtained, such as atorvastatin and thalidomide. In conclusion, UKN1 identifies teratogens with high sensitivity and specificity, and combined interpretation analysis with other tests that differentiate hiPSCs into otherdifferent cell types may further improve the results in the future. Human iPSCs were differentiated for 6 days according to a neuroepithelial protocol (UKN1) and incubated with compounds at the same time. RNA was isolated from these cells and a whole transcritpome analysis was performed with Affymetrix HG-U133 Plus 2.0 arrays. Compound-induced gene expression changes were determined with statistical models.