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BOR - Papers in Press, published online ahead of print March 2, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037150
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Li Wang
Enkui Duan
Li-ying Sung
Byeong-Seon Jeong
Xiangzhong Yang
X. Cindy Tian
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Submitted October 16, 2004
Returned for revision November 16, 2004
Accepted February 16, 2005

Reproductive Technology


Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos

Li Wang , Enkui Duan , Li-ying Sung , Byeong-Seon Jeong , Xiangzhong Yang , and X. Cindy Tian *

* To whom correspondence should be addressed. E-mail: xiuchun.tian{at}uconn.edu.

Abstract
Abstract Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phosphatase (ALPL), SSEA4, and OCT4) that are normally 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 (NT) embryos. These bovine ES cells grew in large, multicellular colonies resembling the mouse ES, 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 ntES cells are 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 over one year, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three embryonic germ 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. This is presumably due to an interruption of the self-renewal pathway. In summary, we generated pluripotent bovine ES cells that have morphology similar to those of the established ES cells in humans and mice and marker staining patterns identical to those of the bovine blastocysts.

Key words: Embryo • In vitro fertilization


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