I-10: The Oocyte Express Way to Reprogramming Supports Double Nucleus Transplantation

Studies on cell fusion-mediated nuclear reprogramming have led to the breakthrough of the induced pluripotent stem (iPS) cell technology. While this technology has neared stem cells to applications more than any other method, the mechanistic bases of reprogramming remain largely unsolved. In this context, comparative studies of oocyte and cell fusion-mediated reprogramming hold the greatest promise to elucidate underlying ground principles. As the endpoints of both methods differ in terms of ploidy of the resultant pluripotent cell lines, we modified the nuclear transfer procedure to neutralize such difference and enable a valid comparison. To this end, instead of a single nucleus, we injected two, or even three nuclei into an enucleated oocyte at the same time. We provide evidence that the oocyte is able to reprogram two somatic nuclei at once and therefore is not limited in its reprogramming capacity to a single nucleus. Tetraploid cloned blastocysts support the derivation of tetraploid NT embryonic stem cells (tNT-ES cells). These cells display the major hallmarks of pluripotency including the ability to differentiate into derivatives of the three germ layers in vitro as well as in vivo. Furthermore these cells are unique in that they embody the first case of cloned ES cells containing two reprogrammed somatic genomes in the same cytoplasm. In sum, the new method of double nucleus transplantation enables the direct comparison of oocyte- and fusion-mediated nuclear reprogramming, thereby providing a useful tool to advance our mechanistic understanding of the reprogramming process.