Factors Affecting the Efficiency of Chimera Production by Coculture of Zona-Free Embryos with Frozen-Thawed Embryonic Stem Cells in Mice.

Zona-free uncompacted 8-cell (8C) or partially compacted 8-cell (8-M) stage embryos were cocultured with frozen-thawed TT2 embryonic stem (ES) cells at a density of either 5.5 × 105 or 5.5 × 106 cells/ml. When zona-free embryos at the 8C and 8-M stage were cocultured with frozen-thawed ES cells at a density of 5.5 × 105 cells/ml, 5% and 32% of the embryos developed to the blastocyst stage after overnight culture, respectively. At a density of 5.5 × 106 cells/ml, however, developmental rates to the blastocyst stage of 8C and 8-M were only 12% and 15%, respectively. When the 8C and 8-M embryos were cocultured with frozen-thawed ES cells at a density of 5.5 × 105 cells/ml, there was no difference in the number of ES cells attached to either of them. The 8-M showed no significant difference in development to blastocyst stage in spite of the number of attached ES cells. However, the development rate to blastocyst was significantly reduced when the 8C aggregated with high numbers of ES cells. When the blastocysts were transferred into recipients, the percentage of chimeric mice in newborns ranged between 0-25%. In comparison, the percentage of newborn chimeric mice after transfer of the morula to small blastocyst ranged between 13-60%. One cause of this high frequency of chimerism after transferring morula derived from 8C was delayed preimplantation development of the embryos with high numbers of attached ES cells.