An alternative method of deriving embryonic stem cell-like clones by aggregation of diploid cells with tetraploid embryos

ObjectiveTo assess whether embryonic stem (ES) cells could be derived from the aggregation of diploid cells with tetraploid embryos.DesignRandomized, prospective study.SettingUniversity embryology and gamete biotechnology laboratory.Animal(s)F1 (C57BL6/DBA2) mice.Intervention(s)Four- to eight-cell F1 tetraploid embryos were aggregated with 10 to 15 donor E14 ES cells.Main Outcome Measure(s)Embryogenesis and ES cell establishment.Result(s)No difference (78% to 89%) in blastocyst formation was detected between the aggregated tetraploid and the control diploid embryos. In a total of 27 transfers, pregnancy was detected in three tetraploid (23.1%) and five diploid (35.7%) cases, and three live births developed from the aggregated tetraploid embryos. The tetraploid blastocysts without aggregation were plated, but no ES cell-like colony was formed. Six of eight aggregated blastocysts derived well-proliferated colonies, which were positive for anti–stage-specific embryonic antigen (SSEA)-1 antibody, Oct-4, and alkaline phosphatase. The microsatellite assay confirmed the homogenous makeup among the donor E14 cells and live-birth and ES-like cells derived from the E14-aggregated, tetraploid embryo.Conclusion(s)The aggregation of pluripotent diploid cells with tetraploid embryos yielded live births and ES-like cells that were homogenous to the donor diploid cells.