Establishment of mouse embryonic stem cell lines from somatic cell nuclei by nuclear transfer into aged, fertilization-failure mouse oocytes

It has been widely assumed that fresh oocytes are required for the establishment of embryonic stem cells (ES cells) via somatic cell nuclear transfer (SCNT). Such cell lines are referred to as ‘NT-ES cells’ [1]. This presumed requirement, however, gives rise to fundamental ethical concerns surrounding its potential application to human cells, as fresh oocytes must be obtained from a healthy female donor. Human in vitro fertilization (IVF) is now performed routinely in infertility clinics, but in the IVF attempts some of the oocytes fail to become fertilized and are discarded [2]. Here we show that such aged fertilization-failure (AFF) oocytes from mice can be successfully used as recipients in nuclear transfer. We suggest that, if recapitulated with human oocytes, this method may provide a means of avoiding some of the ethical concerns surrounding fresh oocyte donation for nuclear transfer. AFF oocytes lacking a second polar body and pronucleus (see Supplemental data published with this article online) were selected at six hours after insemination to serve as recipients for nuclear transfer. The somatic donor cells (cumulus cells) were collected from several mouse strains, including BDF1 — a strain engineered to express green fluorescent protein (GFP) in all body cells. Nuclear transfer and NT-ES cell establishment were performed as described [3,4], with some modifications to timing (Supplemental data). The success rate of nuclear transfer and development to morula or blastocyst stages was significantly lower for AFF oocytes than for fresh oocytes (Figure 1; Table 1). At the two-cell stage, embryos derived from AFF oocytes exhibited small fragments between blastomeres (Figure 1B), which disappeared by the 4or 8-cell stage. However, the rate of establishment of NT-ES cell lines from cloned morulae or blastocyststage embryos was similar for AFF oocytes and fresh oocytes (6% vs. 7%; Table 1; Supplemental data). Surprisingly, even when oocytes from several mouse strains were stored at room temperature for one day (24 h after oocyte collection), they could be used to establish NTES cell lines at similar efficiencies (4%; Table 1; Supplemental data). It is known that oocytes stored at low temperature suffer irreversible damage to spindle structures [5,6]. All established cell lines stained positive for the ES cell-specific markers alkaline phosphatase, Oct3/4 and Nanog, and all randomly selected NT-ES cell lines (n = 13) were of normal karyotype (Supplemental Data). To demonstrate the pluripotency of these NT-ES cell lines, BDF1-GFP derived NT-ES cells were injected into the perivitelline space of either diploid or tetraploid mouse embryos. We obtained a number of healthy and fertile offspring using both methods (Figure 1D; Supplemental data). GFP expression and coat color confirmed that these NT-ES cell lines are truly derived from donor nuclei. Analyses of chimeric contribution showed that these cells are capable of functional differentiation into all cell types required for development and germ cell formation, similar to ES and NT-ES cells derived from fresh oocytes [7]. We additionally examined the full-term developmental potential of cloned embryos and of embryos produced by intracytoplasmic sperm injection (ICSI) using AFF oocytes. After ICSI, AFF oocytes showed much lower rates of development to term than fresh oocytes. In the NT experiments, none of the cloned embryos developed to term when