Deriving bovine embryonic stem-like cells in defined conditions

The first embryonic stem ES cell line was isolated from mouse in 1981 (Evans and Kaufman 1981; Martin1981). However, ESCs are only available in rodent species. Although research has been carried out on bovine embryos for more than two decades, there is no evidence of ESCs (Shanbo, Fang et al. 2009). Reasons for this are unknown developmental aspects associated with bovine embryology. Most attempts to culture bovine ES-like cells have been to isolate them from the ICM of day seven blastocysts, which have been generated by in vitro production (IVP). Here I report an alternative method to derive bES-like cells using dissociated blastomeres from early stages embryos (8 and 16-cell) and day 5 and 7 embryos. These bovine outgrowths were cultured in order to investigate which developmental stage associated with better blastomere attachment. Also, to examine their pluripotency, as it was hypothesised that early stages of development up to the blastocyst stage are pluripotent and that the naive state of bovine ESCs exist. In addition, instead of using animal material as feeder layers such as mouse embryonic fibroblasts (MEFs), which provides leukemic inhibitory factor (LIF) to maintain pluripotency, defined extra cellular matrixes (ECM) were used in order to derive bES-like cells in defined conditions, avoid contamination by reagents used in cell culture and to find the substrate associated with better attachment and proliferation. 2i media was applied to the development of bovine embryos in order to determine if the use of 2i media with different numbers of blastomeres would encourage bES-like cell development, proliferation and to introduce cell uniformity. Blastomeres were cultured singularly, and in groups in 96 well plates and in tissue culture plates in order to produce short term cell lines. Development rate, attachment and outgrowth production were measured, for example, the proportion of blastomeres attached and proliferated. Markers of ESCs, epiblast stem cells (epiESCs) and trophectoderm were investigated in produced bovine outgrowths in order to examine their pluripotency, and their karyotyping was examined. In this study, from the different developmental stages used, outgrowths were produced from all stages used, but the inner cell mass (ICM) was associated with better outgrowth production. Different ECMs promoted attachment, however, with variable efficiency, in which gelatin was associated with better attachment and proliferation. The use of 2i media resulted in attachment, but with poor proliferation. cDNA isolated from bovine outgrowths expressed some of pluripotency markers, but with a lack of uniformity. In addition, metaphase spreads from outgrowths showed an abnormal karyotype. From this study, we increased our understanding of the factors that enhance the derivation of bES-like cells such as the culture media and the substrates. In addition, we have gained more information on bovine gene expression in blastomere derived outgrowths