The Derivation and Characterisation of Bovine Embryonic Stem Cells

Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of preimplantation embryos. ES cells exhibit two common characteristics: the ability to differentiate into all three germ layers, and the ability to self-renew. Scientists first discovered how to derive embryonic stem cells from mouse embryos 40 years ago, in 1981. Almost 20 years later, in 1998, embryonic stem cells were first isolated from humans. Mouse and human embryonic stem cells have been used in a vast array of important research, including regenerative medicine and cancer research. However, the successful isolation and subsequent culture of bovine embryonic cells has proven more difficult. For a long time, embryonic stem-like cells cultured from bovine embryos showed poor derivation efficiencies, limited proliferation, and progressive loss of pluripotency markers. Following decades of attempts, embryonic stem cells were derived from cattle and maintained in a pluripotent state for the first time in 2018. This thesis presents an exploration into the methods used to derive bovine embryonic stem cells, and the culture conditions needed to sustain their self-renewal in a pluripotent state. Outgrowths of the ICM were cultured in conditions which promoted proliferation, and were able to be sustained for upwards of 25 days. Characterisation of these cells using immunohistochemistry and RT-qPCR analysis showed expression of pluripotency related genes, however this expression was limited and appeared to reduce with passaging. The results of this research suggest that bovine ES cells are able to be maintained in similar culture conditions to porcine expanded potential stem cells, however pluripotency is only able to be maintained for a limited number of passages. Consequently, further research is needed in order to fully understand the regulatory mechanisms surrounding pluripotency states in bovine species.