title: Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction.
abstract: Therapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for "therapeutic cloning," though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or "fertilization failures" might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming.
authors.affiliation: Department of Obstetrics-Gynecology-Reproductive Sciences, Pittsburgh Development Center, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Calvin
authors.initials: C
authors.lastname: Simerly
authors.forename: Christopher
authors.initials: C
authors.lastname: Navara
authors.forename: Sang Hwan
authors.initials: SH
authors.lastname: Hyun
authors.forename: Byeong Chun
authors.initials: BC
authors.lastname: Lee
authors.forename: Sung Keun
authors.initials: SK
authors.lastname: Kang
authors.forename: Saverio
authors.initials: S
authors.lastname: Capuano
authors.forename: Gabriella
authors.initials: G
authors.lastname: Gosman
authors.forename: Tanja
authors.initials: T
authors.lastname: Dominko
authors.forename: Kowit-Yu
authors.initials: KY
authors.lastname: Chong
authors.forename: Duane
authors.initials: D
authors.lastname: Compton
authors.forename: Woo Suk
authors.initials: WS
authors.lastname: Hwang
authors.forename: Gerald
authors.initials: G
authors.lastname: Schatten
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D002875:Chromosomes / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods; D003599:Cytoskeleton / Q000378:metabolism*; D004624:Embryo Transfer; D047108:Embryonic Development; D005260:Female; D005306:Fertilization; D006801:Humans; D016547:Kinesins / Q000378:metabolism; D008251:Macaca; D008540:Meiosis / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D017239:Paclitaxel / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000502:physiology
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D020409:Molecular Motor Proteins; D009687:Nuclear Proteins; D016547:Kinesins; D017239:Paclitaxel