Rapid loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines.
The POU transcription factor Oct-4 is essential for the pluripotent character of the mouse inner cell mass (ICM) and derivative embryonic stem (ES) cells. We analyzed the expression of Oct-4 during culture and establishment of cell lines from mouse and rat preimplantation embryos. Oct-4 was rapidly lost in primary outgrowths of the majority of cultured embryos prior to any evidence of morphological differentiation. Oct-4 persisted in only 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 beta-geo is under control of Oct-4 regulatory elements to investigate the effect of direct selection for Oct-4 expressing cells. Ablation of all cells occurred, consistent with complete downregulation of Oct-4. Without selection, in contrast, continuous cultures of morphologically undifferentiated cells could be derived readily from rat blastocysts and ICMs. However, these cells did not express significant Oct-4 and, although capable of differentiating into extraembryonic cell types, appeared incapable of producing fetal germ layer derivatives. Downregulation of Oct-4 appears to be a limiting factor in attempts to derive pluripotent cell lines from preimplantation embryos.
Buehr
M
M
Centre for Genome Research, University of Edinburgh, Edinburgh EH9 3JQ, United Kingdom. mbuehr@srv0.bio.ed.ac.uk
Nichols
J
J
Stenhouse
F
F
Mountford
P
P
Greenhalgh
C J
CJ
Kantachuvesiri
S
S
Brooker
G
G
Mullins
J
J
Smith
A G
AG
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D002678:Chimera / Q000235:genetics; D046508:Culture Techniques; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D017403:In Situ Hybridization; D051379:Mice; D008808:Mice, Inbred CBA; D050814:Octamer Transcription Factor-3; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D014157:Transcription Factors