Abstract
Bovine embryonic stem (bES) cell lines reported to date vary in morphology and marker expression, such as alkaline phosphatase (ALPL), stage-specific embryonic antigen 4 (SSEA4), and octamer-binding transcription factor-4 (OCT4), that normally are associated with the undifferentiated, pluripotent state. This chapter introduces the methods of isolating and maintaining bovine ES cells. These bovine ES cells grow in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells, as well as human EG cells. Throughout the culture period, most of the cells within the colonies stain positive for ALPL and cell surface markers SSEA4 and OCT4. The staining patterns of the bES cells are identical to those of the blastocysts fertilized in vitro (IVF), yet different from most previously reported bovine ES cell lines, which are either negative or not detected. After undifferentiated culture for more than 1 year, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. In addition to bES from IVF, this chapter introduces two methods of generating blastocyst stage embryos other than in vitro fertilization, which are parthenogenetic activation and somatic cell nuclear transfer for the potential application of generation "patient-specific" ES cells.
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