The Derivation and Characterisation of Bovine Expanded Potential Stem Cells

Embryonic stem cells were first derived from the embryo of a mouse in 1981. They are characterised by their ability to eternally self-renew in proper conditions in cell culture, and their ability to be directed to differentiate into all adult tissue types. In the last 40 years embryonic stem cells have been derived in mice, then humans and more recently in livestock animals including cattle, pigs, and horses. Stem cells have been used in a wide range of research areas, with more recent developments of 3D cell cultures and organoids being used in complex modelling of diseases like cancer and Covid-19. This thesis explores a new method for the derivation of expanded potential stem cells first used to derive mouse expanded potential stem cells, and subsequently applied to derive human and porcine expanded potential stem cells. Hatched bovine blastocyst-stage embryos were plated on mouse embryonic fibroblast feeders using a chemically defined media, which promoted outgrowth of the inner cell mass. Outgrowths were able to be passaged twice, surviving up to 20 days. Immunohistochemistry and alkaline phosphatase staining showed limited expression of pluripotency related genes, and no expression of trophectoderm genes. However, after passaging, much of this expression was lost. While the general principles of the process show promise, further research is needed to optimise the process of derivation and improve passaging quality to properly derive a bovine embryonic stem cell line.