Blastocyst formation, karyotype, and mitochondrial DNA of interspecies embryos derived from nuclear transfer of human cord fibroblasts into enucleated bovine oocytes

Objective: To establish an interspecies somatic cell nuclear transfer (iSCNT) technique for deriving blasto-cysts having human chromosome complements without sacrificing human oocytes. Design: Prospective, randomized study undertaken in vitro. Setting: University-affiliated hospital and laboratory, Seoul National University. Patient(s): Postpartum women with natural spontaneous vaginal delivery. Intervention(s): Human cord fibroblasts were retrieved from five postpartum women from whom informed consent was obtained. After subculture and cryopreservation, serum-starved cells were transferred into enucleated bovine oocytes. Main Outcome Measure(s): Embryo development, karyotype, and the presence of mitochondrial DNA (mtDNA). Result(s): A total 1,742 oocytes were provided for iSCNT and results showed that both fibroblast batch and reconstruction method significantly affected iSCNT outcome. An iSCNT using a single DC pulse of 1.9-2.1 kV/cm for 20 microseconds yielded better rates of fusion (30%-56%) and cleavage (36%) than the other iSCNT protocols. Four to 9% interspecies embryos produced with the optimized method developed to morulae or blastocysts after cultured in a serum-free medium. Results from karyotyping demonstrated that 56% of interspecies embryos evaluated had human chromosome complements. In polymerase chain reaction (PCR) analysis of a single embryo, both human and bovine rmDNAs were detected until the 16-cell stage, whereas only the bovine mtDNA was found beyond the morula stage. Conclusion(s): An iSCNT using human cord fibroblasts and bovine oocytes can yield blastocysts and the results of karyotyping and mtDNA analysis confirmed the feasibility of the iSCNT technique. (C) 2003 by American Society for Reproductive Medicine.