Generation and characterization of pluripotent stem cells from cloned bovine embryos.
Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phosphatase [ALPL], stage-specific embryonic antigen 4 [SSEA4], and OCT4) that normally are associated with the undifferentiated, pluripotent state. These observations suggest that the proper experimental 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 colonies 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 nuclear 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 pluripotency. However, unlike the mouse and human ES cells, following treatment with trypsin, type IV collagenase, or protease E, our bovine ES cells failed to self-renew and became spontaneously differentiated. Presumably, this resulted from an interruption 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-staining patterns identical to those of the bovine blastocysts.
Wang
Li
L
Institute of Zoology, Chinese Academy of Sciences, Beijing.
Duan
Enkui
E
Sung
Li-ying
LY
Jeong
Byeong-Seon
BS
Yang
Xiangzhong
X
Tian
X Cindy
XC
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002454:Cell Differentiation / Q000502:physiology*; D019976:Cloning, Organism / Q000662:veterinary*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D006028:Glycosphingolipids / Q000378:metabolism; D007150:Immunohistochemistry / Q000662:veterinary; D007621:Karyotyping / Q000662:veterinary; D008297:Male; D050814:Octamer Transcription Factor-3; D010312:Parthenogenesis; D039904:Pluripotent Stem Cells / Q000502:physiology* / Q000648:ultrastructure; D011247:Pregnancy; D055746:Stage-Specific Embryonic Antigens; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; D006028:Glycosphingolipids; D050814:Octamer Transcription Factor-3; D055746:Stage-Specific Embryonic Antigens; D014157:Transcription Factors; C066584:stage-specific embryonic antigen-4; D000469:Alkaline Phosphatase