title: Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts.
abstract: Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20-24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer.
authors.affiliation: Stemagen Corporation, 4150 Regents Park Row, La Jolla, California 92037, USA. afrench@stemagen.com
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: French
authors.forename: Catharine A
authors.initials: CA
authors.lastname: Adams
authors.forename: Linda S
authors.initials: LS
authors.lastname: Anderson
authors.forename: John R
authors.initials: JR
authors.lastname: Kitchen
authors.forename: Marcus R
authors.initials: MR
authors.lastname: Hughes
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wood
mesh_terms: D000328:Adult; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism; D002460:Cell Line; D019976:Cloning, Organism; D004272:DNA, Mitochondrial / Q000235:genetics; D046149:Embryo Culture Techniques; D047108:Embryonic Development; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010312:Parthenogenesis
chemical_list: D004272:DNA, Mitochondrial