title: Improvement in the blastocyst quality and efficiency of putative embryonic stem cell line derivation from porcine embryos produced in vitro using a novel culturing system.
abstract: Porcine embryonic stem cells (pESCs) have great potential for application in translational biomedical research, including xenotransplantation and disease models. Obtaining high-quality blastocysts is the most important factor in the isolation and colonization of primary ESCs and the establishment of ESC lines. In pigs, in vitro-derived blastocysts have a limited cell number compared to in vivo-derived blastocysts and show an indefinite inner cell mass, which may result in failure to establish pESC lines. In the present study, the effects of resveratrol (RES), granulocyte-macrophage colony stimulating factor (GM-CSF) and β-mercaptoethanol (β-ME) on the quality of blastocysts and the efficiency of colony derivation were investigated for the establishment of ESCs. A novel culturing system was developed in which 2 µM RES was added to the oocyte in vitro maturation (IVM) medium, and 10 ng/ml pGM-CSF and 10 µM β-ME were added to embryo in vitro culture (IVC) medium. This novel system showed significantly more parthenogenetic activation (PA) blastocysts (54.5 ± 1.8% vs. 43.4 ± 1.2%; P<0.05) and in vitro fertilization (IVF) blastocysts (36.9 ± 3.3% vs. 26.2 ± 2.9%; P<0.06) at day seven as compared with that in the control system. The PA and IVF blastocysts from the novel system showed a significantly greater hatching rate (P<0.05) and greater cell numbers (55.1 ± 2.0 vs. 45.6 ± 2.0; P<0.05 and 78.9 ± 6.8 vs. 58.5 ± 7.2; P<0.06, for PA and IVF, respectively) at day seven compared to that in the control system. After seeding on feeder cells, the PA blastocysts produced by the novel system showed a significantly increased rate of attachment (28.8 ± 3.9% vs. 17.2 ± 2.4%; P<0.062). Finally, two putative pESC lines from PA embryos produced by the novel system and one by the control system were established. In conclusion, the novel system improved blastocyst quality and increased the derivation efficiency of putative pESC lines from porcine PA and IVF embryos produced in vitro.
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Seung A
authors.initials: SA
authors.lastname: Cheong
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Eunhye
authors.initials: E
authors.lastname: Kim
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Seong-Sung
authors.initials: SS
authors.lastname: Kwak
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Yubyeol
authors.initials: Y
authors.lastname: Jeon
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Sang-Hwan
authors.initials: SH
authors.lastname: Hyun
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D020869:Gene Expression Profiling; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008297:Male; D008623:Mercaptoethanol / Q000378:metabolism; D051379:Mice; D010312:Parthenogenesis; D000077185:Resveratrol; D013267:Stilbenes / Q000378:metabolism; D013552:Swine / Q000196:embryology*
chemical_list: D013267:Stilbenes; D008623:Mercaptoethanol; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000077185:Resveratrol