ES cell lines from tetraploid mouse blastocysts

Abstract

Testing of mouse stem cells for pluripotency is mainly conducted with either of three different assays: embryoid body (EB) formation, teratoma formation, and tetraploid (4 N) embryo complementation. The complementation assay relies on the assumption that the 4 N embryo lacks in own pluripotency. Here we devised a new method for generating 4 N mouse embryos and, from these embryos, we derived functional 4 N embryonic stem (ES) cells. Our method uses the nuclear transplantation (NT) of two somatic cell nuclei (diploid, 2 N) into oocytes deprived of their metaphase II spindle (ooplasts), or the intracytoplasmic sperm injection (ICSI) of two sperm heads into intact oocytes. It follows that these embryos are 4 N from the beginning of embryonic life, in contrast to conventional methods that use the fusion of two 2 N embryo blastomeres. The derivation of 4 N ES cells proves that the inner cell mass (ICM) of a mouse 4 N blastocyst is pluripotent. Here we apply this new method to determine whether one mouse ooplasm can reprogram two somatic nuclei, and to separate the effects of chromosome duplication and centriole duplication from each other in the process of tetraploidization that can lead to tumor formation.