Accuracy, discriminative properties and reliability of a human ESC-based in vitro toxicity assay to distinguish teratogens responsible for neural tube defects

Abstract

The poor correlation of developmental toxicity studies in animals with human outcome data has emphasized the need for complementary assays based on human cells and tissues. As neural tube defects represent an important proportion of congenital malformations, we evaluated here the accuracy of a human embryonic stem cell (hESC)-based assay to predict chemically induced disruption of neural tube formation. As teratogenic compounds, we used cyclopamine (CPA), valproic acid (VPA), ochratoxin A (OTA) and mycophenolic acid (MMF), all suspected or known inducers of human neural tube defects, as well as theophylline and saccharin as negative control compounds. We analyzed their effects on the ability of hES cells to give rise to neural precursors (expressing specific marker Nestin), to form neural tube-like structures (rosettes), and to express specific markers (Sox1, Otx2, Lix1, EvI1, Rspo3) during rosette formation. The results showed that various effects of the selected compounds on early neural development could be specifically revealed in vitro through related alterations of neurogenic differentiation of hESC. Furthermore, it was possible to discriminate toxicants acting at different time points during embryonic development and, therefore, responsible for distinct adverse effects on neural tube formation. By comparing four different hESC lines, we observed a significant (up to fivefold) variability of the line-dependent response to toxicants. We highlight at least two sources of variability: one related to the heterogeneity of hESC lines in culture (stemness/commitment profiles); the second to possible genetically determined differences in individual sensitivity to teratogens.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

An efficient neuron-astrocyte differentiation protocol from human embryonic stem cell-derived neural progenitors to assess chemical-induced developmental neurotoxicity

“…After EB formation, individual EBs grew out into neural rosettes over the course of one week (day 5-12). In order to obtain a more homogeneous NPC culture, neural rosettes were selected and replated to grow as rosettes for a second time (day [12][13][14][15][16][17][18][19]. This step was repeated on day 19 and cells were grown as NPCs for one week (day 19-26) before further expansion to freeze down the cells or start neuronastrocyte generation cultures.…”

“…Functional neuronal network development in vitro has proved challenging as spontaneous electrical activity was difficult to measure and/ or differentiation took several weeks [13][14][15][16][17][18]. These drawbacks represent important limitations for implementation in regulatory testing.…”