Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

• Published in: Toxicology in vitro 2015-04, Vol.29 (3), p.449-457
• Main Authors: Schulpen, Sjors H.W., de Jong, Esther, de la Fonteyne, Liset J.J., de Klerk, Arja, Piersma, Aldert H.
• Format: Article
• Language: English
• Subjects: Animals; Anticonvulsants - pharmacology; Carbamazepine; Carbamazepine - toxicity; Cell Differentiation - drug effects; Embryonic Stem Cells - drug effects; Embryonic Stem Cells - metabolism; Fibroblasts; Gene Expression - drug effects; Human embryonic stem cells; Humans; Mice; Neural differentiation; Neurodevelopmental toxicity; Neurons - drug effects; RNA - biosynthesis; Valproic acid; Valproic Acid - toxicity
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2014.12.001

•Designed a practical 11day protocol for hESC differentiation into neural cells.•Time-dependent down regulation of stem cell related genes.•Time-dependent increasing expression of neurogenesis involved genes.•7Days exposure to increasing concentrations VPA and CBZ caused concentration dependent effects.•Demonstrated effectiveness of this assay to study mechanism involved in developmental toxicity. Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as βIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of βIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.