PARENT SESSION
Preimplantation Embryo/Fetal Development

DNA METHYLTRANSFERASE 1 IS ESSENTIAL FOR ESTABLISHMENT OF TROPHOBLAST STEM CELLS IN CULTURE.

Oda, Mayumi¹, Tanaka, Satoshi¹, Shiota, Kunio¹, ¹

ABSTRACT- DNA methylation patterns in mammals are stage- and tissue-specific. This suggests that methylation patterns contribute to proper cell differentiation and/or to the maintenance cellular characteristics. Several different DNA methyltransferases exist that both establish and maintain proper methylation patterns. Dnmt1 is largely a maintenance methyltransferase that ensures an established methylation pattern is inherited by daughter cells. Disruption of Dnmt1 causes growth delay at gastrulation and homozygous mutants are embryonic lethal. To evaluate the role of Dnmt1 in placental development, we attempted to establish trophoblast stem (TS) cells from homozygous Dnmt1 hypomorphic mutant embryos. TS cells exclusively contribute to the trophoblast lineage in vivo in chimeras. At the blastocyst stage, TS cells appear in the polar trophectoderm, which is adjacent to the inner cell mass. TS cells can be maintained in culture with media supplemented with FGF-4, and differentiate into several subtypes of trophoblast cells. To establish homozygous mutant TS cells, we used blastocysts from heterozygous matings. The 39 blastocysts from 6 female mice were incubated in FGF4-contained medium with feeder cells. Of 39 blastocysts, 28 generated stem cell colonies; 3 were homozygous, 10 were wild type, 13 were heterozygous clones, and 2 could not be genotyped, but were probably also homozygous. All clones were passed more two times and most (70-80%) of wild type and heterozygous clones survived. However, no homozygous clone survived beyond the second passage. Some autonomous differentiation was observed in the homozygous TS cells. Our results indicate that Dnmt1-hypomorphic TS cells cannot be established in cell culture, as normal and heterozygous TS cells can. This is in contrast to Dnmt1-hypomorphic embryonic stem (ES) cells, which can be maintained in culture. These data suggest that the trophoblast lineage is more dependent on Dnmt1 than the embryo proper.

KEY WORDS: mouse, DNA methyltransferase, TS cell