Generation and Characterization of Pluripotent Stem Cells from Cloned

Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phospha-tase [ALPL], stage-specific embryonic antigen 4 [SSEA4], and OCT4) that normally are associated with the undifferentiated, pluripotent state. These observations suggest that the proper ex-perimental conditions for consistently producing bovine ES cells have not been identified. Here, we report three bovine ES cell lines, one from in vitro-fertilized and two from nuclear transfer embryos. These bovine ES cells grew in large, multicellular col-onies resembling the mouse ES and embryonic germ (EG) cells and human EG cells. Throughout the culture period, most of the cells within the colonies stained positive for ALPL and the cell surface markers SSEA4 and OCT4. The staining patterns of nu-clear transfer ES cells were identical to those of the blastocysts generated in vitro yet different from most previously reported bovine ES cell lines, which were either negative or not detected. After undifferentiated culture for more than 1 yr, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their plu-ripotency. However, unlike the mouse and human ES cells, fol-lowing treatment with trypsin, type IV collagenase, or protease E, our bovine ES cells failed to self-renew and became sponta-neously differentiated. Presumably, this resulted from an inter-ruption of the self-renewal pathway. In summary, we generated pluripotent bovine ES cells with morphology similar to those of established ES cells in humans and mice as well as marker-stain-ing patterns identical to those of the bovine blastocysts. embryo, in vitro fertilizatio