26 REPROGRAMMING OF TWO SOMATIC NUCLEI IN THE SAME MOUSE OOPLASM LEADS TO PLURIPOTENT NOT TOTIPOTENT EMBRYOS

Abstract

The use of cloned embryos to derive pluripotent embryonic stem cells (ESC) has raised expectations among many for its potential to benefit regenerative medicine. However, diploid cloned embryos also have the ability for totipotency and they occasionally develop to term. Thus, a therapeutic application cannot be kept totally separate from reproductive cloning. In contrast to diploidy, tetraploidy is incompatible with full development in mammals; yet, tetraploid cells arise after normal diploid development and contribute to certain organs of the adult body. In this context, we asked if single oocytes are able to reprogram 2 simultaneously transplanted somatic nuclei, and whether resultant embryos may serve as a source for functional pluripotent cell lines. We modified the conventional nuclear cloning method by simultaneously injecting 2 nuclei of cumulus cells into a single ooplasm of the B6C3F1 mouse strain. Following activation of these reconstructed oocytes, the blastocyst rates were lower compared with conventional cloned embryos (1 nucleus injection), but similar to tetraploid fertilized embryos generated by intracytoplasmic sperm injection (ICSI) of 2 sperm heads and prevention of second polar body extrusion using cytochalasin B. Upon transfer of tetraploid cloned blastocysts to pseudopregnant recipients, no fetuses were found at midgestation (Table 1). Tetraploid cloned blastocysts supported the derivation of ESC (Table 1), which maintain a stable tetraploid karyotype over more than 20 passages. These tetraploid ESC possess the major hallmarks of pluripotency, including matching gene expression profiles (MouseRef-8 v2 expression BeadChip, Illumina) compared with those of diploid counterparts (>99% similarity) as well as the ability to differentiate into derivatives of the 3 germ layers when forming teratomas in severe-combined immunodeficient mice. Upon injection into wild-type blastocysts, tetraploid ESC populated the inner cell mass; however, after transfer of these chimeras to pseudopregnant recipients, derivatives of tetraploid ESC were found only in extraembryonic tissues of midgestation fetuses. In sum, we have shown that the reprogramming capacity of mouse oocytes is sufficient for 2 somatic nuclei, and that resultant cloned embryos support pluripotency but not totipotency. Our approach to therapeutic cloning is free of reproductive implications. Table 1.Developmental competence of single (2N) and double nucleus transplanted (4N) cloned mouse embryos compared with fertilized controls