title: Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos.
abstract: Nuclear transfer (NT) from porcine iPSC to create cloned piglets is unusually inefficient. Here we examined whether such failure might be related to the cell cycle stage of donor nuclei. Porcine iPSC, derived here from the inner cell mass of blastocysts, have a prolonged S phase and are highly sensitive to drugs normally used for synchronization. However, a double-blocking procedure with 0.3 μM aphidicolin for 10 h followed by 20 ng/ml nocodazole for 4 h arrested 94.3% of the cells at G2/M and, after release from the block, provided 70.1% cells in the subsequent G1 phase without causing any significant loss of cell viability or pluripotent phenotype. Nuclei from different cell cycle stages were used as donors for NT to in vitro-matured metaphase II oocytes. G2/M nuclei were more efficient than either G1 and S stage nuclei in undergoing first cleavage and in producing blastocysts, but all groups had a high incidence of chromosomal/nuclear abnormalities at 2 h and 6 h compared with non-synchronized NT controls from fetal fibroblasts. Many G2 embryos extruded a pseudo-second polar body soon after NT and, at blastocyst, tended to be either polyploid or diploid. By contrast, the few G1 blastocysts that developed were usually mosaic or aneuploid. The poor developmental potential of G1 nuclei may relate to lack of a G1/S check point, as the cells become active in DNA synthesis shortly after exit from mitosis. Together, these data provide at least a partial explanation for the almost complete failure to produce cloned piglets from piPSC.
authors.affiliation: Division of Animal Sciences and Bond Life Sciences Center; University of Missouri; Columbia, MO USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Division of Animal Sciences; University of Missouri; Columbia, MO USA.
authors.forename: Kiho
authors.initials: K
authors.lastname: Lee
authors.affiliation: Division of Animal Sciences; University of Missouri; Columbia, MO USA; Department of Animal Science and Technology; Sunchon National University; Suncheon, Jeonnam, Korea.
authors.forename: Kwang-Wook
authors.initials: KW
authors.lastname: Park
authors.affiliation: Division of Animal Sciences; University of Missouri; Columbia, MO USA.
authors.forename: Lee D
authors.initials: LD
authors.lastname: Spate
authors.affiliation: Division of Animal Sciences; University of Missouri; Columbia, MO USA.
authors.forename: Randall S
authors.initials: RS
authors.lastname: Prather
authors.affiliation: Division of Animal Sciences; University of Missouri; Columbia, MO USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Wells
authors.affiliation: Division of Animal Sciences and Bond Life Sciences Center; University of Missouri; Columbia, MO USA.
authors.forename: R Michael
authors.initials: RM
authors.lastname: Roberts
mesh_terms: D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods*; D004247:DNA / Q000096:biosynthesis; D004622:Embryo, Mammalian / Q000196:embryology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D053652:Nuclear Transfer Techniques; D011003:Ploidies; D053606:Receptors, OSM-LIF / Q000378:metabolism; D013552:Swine; D046509:Tissue Culture Techniques
chemical_list: D004791:Enzyme Inhibitors; D053606:Receptors, OSM-LIF; D016590:Aphidicolin; D004247:DNA; D008148:Lovastatin; D015739:Nocodazole
keywords: G1 check point; cell cycle; induced pluripotent stem cell; nuclear transfer; porcine