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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.006197
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Submitted April 2, 2002
Returned for revision April 30, 2002
Accepted July 1, 2002

Embryo


Rapid Loss of Oct-4 and Pluripotency in Cultured Rodent Blastocysts and Derivative Cell Lines

M. Buehr 1*, F. Stenhouse 1, J. Nichols 2, P. Mountford 3, C. J. Greenhalgh 4, S. Kantachuvesiri 1, G. Brooker 1, J. Mullins 1, A. G. Smith 1
1 University of Edinburgh
2 University of Edinburgh
3 Stem Cell Sciences Ltd.
4 Royal Melbourne Hospital

* To whom correspondence should be addressed. E-mail: mbuehr{at}srv0.bio.ed.ac.uk.

Abstract

The POU transcription factor Oct-4 is essential for the pluripotent character of the mouse inner cell mass and derivative embryonic stem (ES) cells. Here we have analysed the expression of Oct-4 during culture and establishment of cell lines from mouse and rat pre-implantation embryos. We report that Oct-4 is rapidly lost in primary outgrowths of the majority of cultured embryos, prior to any evidence of morphological differentiation. Oct-4 persists only in a minority of strain 129 cultures, which can go on to give ES cells. We used transgenic rats in which the dual reporter/selection marker ßgeo is under control of Oct-4 regulatory elements to investigate the effect of direct selection for Oct-4 expressing cells. This resulted in ablation of all cells, consistent with complete down-regulation of Oct-4. Without selection, in contrast, continuous cultures of morphologically undifferentiated cells could readily be derived from rat blastocysts and ICMs. However, these cells do not express significant Oct-4 and although capable of differentiating into extraembryonic cell types appear incapable of producing foetal germ layer derivatives. We conclude that down-regulation of Oct-4 is a limiting factor in attempts to derive pluripotent cell lines from pre-implantation embryos.



Key words: Embryo • Developmental biology • Early development • Gene regulation


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