303 THE MODIFIED CULTURE SYSTEM OF PORCINE IN VITRO PRODUCTION IMPROVES COLONIZATION OF PUTATIVE EMBRYONIC STEM CELLS

Abstract

The establishment of porcine embryonic stem cells (pESC) using in vitro-produced blastocysts derived from IVF, parthenogenesis (PA), and somatic cell nuclear transfer (SCNT) have a great potential. However, porcine in vitro-produced blastocysts show a lower quality than do in vivo-derived blastocysts. In this study, to improve in vitro-derived blastocyst quality, and then to establish pESC, we treated IVF embryos and PA embryos with resveratrol (RES), porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), and β-mercaptoethanol (β-ME). The control system was produced using M199 medium in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The modified group was produced using M199 with 2 µM RES in IVM and PZM5 with 10 ng mL–1 pGM-CSF, 2 µM RES, and 10 µM β-ME in IVC. Data were analysed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test. In total, 1210 PA, 612 IVF, and 5 SCNT embryos were evaluated (for SCNT, we examined only the control system). We observed that overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (P < 0.05) in the modified system (54.5%) compared with the control system (43.4%) in PA and hatched blastocysts rates at Day 6 and 7 were also increased significantly. Total blastocyst cell numbers were significantly higher (P < 0.05) in the modified system (55.1) compared with the control system (45.6). Hatching rate of Day 7 IVF blastocysts was also significantly increased. After seeding porcine blastocysts, the attachment rates were higher in the modified system (32.2% in PA and 36.2% in IVF; not examined in SCNT) than the control system (19.5% in PA, 26.6% in IVF, and 40.0% in SCNT). In addition, colonization rates and cell line derivation rates were higher in the modified system than in the control system. Colonization rates of modified system were 13.1% for PA and 17.75% for IVF embryos, whereas the control system generated 2.4% for PA, 10.8% for IVF, and 20.0% for SCNT. We also investigated the correlation between blastocyst state and attachment rate. The highest attachment rate is in hatched blastocyst (78.35 ± 15.74%). Therefore, the modified system increased quality of porcine blastocysts in vitro produced, and subsequently increased attachment rates. The cell line derivation rates were 2.4% (PA), 4.2% (IVF), and 20.0% (SCNT) in control group. In modified system, they were 7.2% (PA) and 10.0% (IVF). We established 3 cell lines from PA blastocysts (1 cell line in control system and 2 cell lines in modified system) and 1 cell line from SCNT-control system. All cell lines showed alkaline phosphatase activity and expressed pluripotent markers. In conclusion, the modified system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of in vitro-produced porcine blastocysts, and subsequently increased derivation rates of putative pESC. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (#2012004885).