Date of Award:

5-2013

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Animal, Dairy, and Veterinary Sciences

Advisor/Chair:

Lee F. Rickords

Abstract

Embryonic stem cells are pluripotent cells isolated from morula stage embryos or the inner cell mass of blastocyst stage embryos. They are capable of differentiating into tissues of all three primary germ layers. In recent years pluripotent cell lines have been created from somatic cell types using various methods, the primary method being viral transduction of exogenous Oct4, Sox2, Klf4, and c-Myc or Oct4, Sox2, Nanog, and Lin28 transgene constructs. The resulting cell lines are termed induced pluripotency stem cells, and are similar to embryonic stem cells in many ways. However, these cell lines are not acceptable for clinical applications due to the use of both modified viral vectors and insertion of exogenous transgenes in their production. Recently the small molecule RepSox, a TGF-ß pathway inhibitor, was used to replace Sox2 during cellular reprogramming of murine embryonic fibroblasts. We evaluated the effects of RepSox on expression of pathways related to pluripotency in murine embryonic fibroblast, murine embryonic stem, and bovine embryonic fibroblast cells. Each cell type was treated with RepSox for 72 hours and subjected to standard qPCR for gene expression analysis. PCR arrays specific to stem cell pathways were used to initially evaluate the effects of RepSox on candidate genes. A subset of genes was then selected for further analysis based on these initial results. We report that RepSox inhibition of the TGF-ß pathway in murine embryonic fibroblasts results in significant upregulation of components of the Wnt, Notch, and Hedgehog signaling pathways, all of which have been linked to stem cell maintenance. In addition, we observed significant upregulation of genes associated with embryonic, mesenchymal, stem cell, and neural cell lineages, indicating that RepSox may be useful in direct reprogramming of murine cells to other somatic cell types. RepSox treatment of murine embryonic stem cells did not result in consistent upregulation of Wnt, Notch, or Hedgehog pathway components, but did result in upregulation of Sox2 and Klf4 expression. Lastly, RepSox treatment of bovine embryonic fibroblasts did not result in the same effects as seen in murine fibroblasts, indicating a need for further analysis to determine the effects of RepSox on bovine cells.

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