64 RECONSTRUCTION OF HETEROGENEOUS EMBRYOS BY HUMAN SOMATIC CELLS AND BOVINE ENUCLEATED OOCYTES AND ISOLATION OF PUTATIVE HUMAN EMBRYONIC STEM CELL CLONES

This study was undertaken to reconstruct heterogeneous nuclear-transferred embryos by using human fetal skin fibroblast cells as nuclear donor cells and the enucleated bovine oocytes as recipient cytoplasts for the purpose of investigating the feasibility of enucleated bovine oocyte cytoplasm as a means of reprogramming human somatic cell nuclei in an attempt to generate an accessible, autologous, and potentially unlimited source of totipotent human embryonic stem cells for transplantation medicine. Bovine ovaries were recovered at a local abattoir and oocytes were in vitro-matured and employed as recipient cytoplasts. A human fibroblast cell line was derived from an aborted fetus at 4 months of age, serum-starved, and used as donor somatic cells. The cultured nuclear transfer embryos were visually assessed for the first completion of cleavage at 48 h of culture, and for subsequent developmental stages at 72 to 168 h. The fusion of fibroblast cells into recipient cytoplasm was induced by electroporation. The fused oocytes were activated by ionomycin with 2 m mL–1 6-DMAP. The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% (v/v) fetal calf serum (FCS) for 168 h. Morulae and blastocysts were used for isolating embryonic stem cells. The results indicated that human fetal skin fibroblast cells could maintain normal morphology and characteristics in culture conditions. They proliferated constantly and presented a regular growth curve in culture. Of these cells, 83.3% retained normal numbers of chromosomes after over 20 culture passages. The skin fibroblast cells exhibited normal morphology and retained normal numbers of chromosomes (2n = 64) in serum starvation culture after undergoing freezing and thawing treatment. The first completed cleavage of xenonuclear transplantation human embryos occurred between 24 and 48 h after activation, and morula and blastocyst development was completed between 72 and 168 h. The cleavage rate and the percentage of blastocyst development of the reconstructed embryos were 80.0% and 7.5%, respectively. Putative embryonic stem cell clones were observed, with nest-like morphology, after 3–7 days of culture on a fibroblast cell layer. Identifications by alkaline phosphatase (AKP) showed that the clones presented a positive reaction, which demonstrated that the isolated stem cell clones were embryonic stem cells. This study demonstrated that xenonuclear-transferred human embryos can undergo embryonic division and subsequent development to morula and blastocyst stage, and that human fetal fibroblast nuclei can be reprogrammed in bovine enucleated oocytes. Xenonuclear-transferred human embryos can be an alternative for obtaining human embryonic stem cells.