Developmental toxicology in vitro using serum-free and feeder-free culture of embryonic stem cells
Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
The development from a fertilized oocyte to an individual containing more than a hundred different types of cells. The embryonic development is one of the most complex biological process and is therefore susceptible to interference. Pharmaceuticals have the ability to interfere with embryonic development, and it is important to assess their developmental toxicity potential. Testing for developmental toxicity requires a large number of animals, and is associated with high costs and ethical concern. The needs for inexpensive and simple assays have resulted in stem cell-based test systems like the embryonic stem cell test (EST). Conventionally grown embryonic stem cells (ESCs) need Leukemia Inhibitory Factor (LIF), bovine serum, and feeder cells (primary fibroblast cells from mouse embryos). The use of serum and feeder cells causes ethical concerns, and problems with variation between batches. In this study conventionally cultured R1 mouse ESCs are adapted to the serum- and feeder- free ESGRO CompleteTM system, and are evaluated by studying gene expression after a short- time (6 hours) exposure to teratogenic and non-teratogenic drugs such as valproic acid (VPA), teratogenic and non-teratogenic VPA analogs with or without the ability to inhibit the enzyme histone deacetylase (HDAC). HDAC is a possible molecular target for VPA, and also the HDAC inhibitor trichostatin A (TSA). Expression of the stemness marker genes (Nanog, Pou5f and Sox2) in the ESGRO-adapted cells indicated that the cells were still pluripotent, and they were therefore used for further experiments. Gene expression studies after exposure of VPA showed a concentration-dependent downregulation of Cyp26a1 and Gsn, and an upregulation of Hap1 and Yap1. This is consistent with previous results in conventionally cultured ESCs. Neither the non-teratogenic VPA analog nor the teratogenic VPA analog that is an HDAC-non inhibitor gave a significant response when the gene expression was compared against the vehicle (0.1% DMSO), whereas the VPA analog that is an HDAC inhibitor gave a significant response for all the studied genes. TSA affected the gene expression of the genes Hap1 and Gsn, but not Cyp26a1 and Yap1, which might indicate that the expression of Hap1 and Gsn is HDAC dependent. The results from this study show the potential of this feeder- and serum-free system as a mechanism-based assay for developmental toxicity of drugs structurally related to VPA. But further identification of genes linked to a wide range of known human teratogens needs to be included to this system to become a tool in the drug development.
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IdentifiersURN: urn:nbn:se:uu:diva-209916OAI: oai:DiVA.org:uu-209916DiVA: diva2:659909