Pluripotent Stem Cells of Embryonic Origin : Applications in Developmental Toxicology

Sammanfattning: General toxicity evaluation and risk assessment for human exposure is essential when developing new pharmaceuticals and chemicals. Developmental toxicology is an important part of this risk assessment which consumes large resources and many laboratory animals. The prediction of developmental toxicity could potentially be assessed in vitro using embryo-derived pluripotent stem cells for lead characterization and optimization. This thesis explored the potential of short-time assays with pluripotent stem cells of embryonic origin using toxicogenomics. Three established pluripotent stem cell lines; P19 mouse embryonal carcinoma (EC) cells, R1 mouse embryonic stem (mES) cells, and SA002 human embryonic stem (hES) cells were used in the studies. Valproic acid (VPA), an antiepileptic drug which can cause the neural tube defects spina bifida in human and exencephaly in mouse, was used together with microarrays to investigate the global transcriptional response in pluripotent stem cells using short-time exposures (1.5 - 24 h). In addition to VPA, three closely related VPA analogs were tested, one of which was not teratogenic in mice. These analogs also differed in their ability to inhibit histone deacetylase (HDAC) allowing this potential mechanism of VPA teratogenicity to be investigated. The results in EC cells indicated a large number of genes to be putative VPA targets, many of which are known to be involved in neural tube morphogenesis. When compared with data generated in mouse embryos, a number of genes emerged as candidate in vitro markers of VPA-induced teratogenicity. VPA and its teratogenic HDAC inhibiting analog induced major and often overlapping deregulation of genes in mES cells and hES cells. On the other hand, the two non-HDAC inhibiting analogs (one teratogenic and one not) had only minor effects on gene expression. This indicated that HDAC inhibition is likely to be the major mechanism of gene deregulation induced by VPA. In addition, a comparison between human and mouse ES cells revealed an overlap of deregulated genes as well as species specific deregulated genes.