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

Objective: To assess whether embryonic stem (ES) cells could be derived from the aggregation of diploid cells with tetraploid embryos. Design: Randomized, prospective study. Setting: University 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