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 blastocysts 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 mtDNAs 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.