돼지 유사 배아줄기세포의 집락 형성률에서 새로운 체외 배아 배양 시스템의 영향 = Effect of Novel in vitro Culture System on Colonization of Putative Porcine Embryonic Stem Cells

다국어 초록 (Multilingual Abstract)

Genetically modified pigs have a great potential as organ donors for xenotransplantation, as well as a model for human diseases, and the porcine embryonic stem cells (pESCs) could be also applied to the translational medicine for the disease models. On their purpose, many researchers have tried to establish pESCs using in vitro produced blastocysts such as in vitro fertilization (IVF) and parthenogenesis (PA). However, in vitro derived porcine blastocyst is no obvious inner cell mass (ICM) or they eventually contain only few cells. Therefore, to improve in vitro derived blastocyst’s quality and then establish pESCs, I investigated the relationship in vitro produced blastocyst quality and colonization efficiency of pESCs. In experiment, the control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The novel system is produced using M199 with 2 μM resveratrol (RES) in IVM and PZM5 with 10 ng/mL porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), 2 μM RES and 10 μM β-mercaptoethanol (β-ME) in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test or Student T test. As results, overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (P < 0.05) in the novel system (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (P <0.05) in the novel system (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the novel system (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in novel system than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but novel system were 17.75% in IVF, and 13.1% in PA. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In novel system, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in novel system). All cell line has alkaline phosphatase activity and express pluripotent markers and differentiation markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• Ⅱ. MATERIALS AND METHODS 3
• 1. Chemicals 3
• 2. In vitro maturation (IVM) of porcine oocytes 3
• 3. In vitro fertilization (IVF) of porcine oocytes 4
• 4. Parthenogenetic activation (PA) of oocytes 4
• 5. Embryo evaluation and total cell count 5
• 6. Gene expression analysis of blastocysts and putative pESCs by real-time PCR or reverse transcriptase PCR 6
• 7. Preparation of mouse embryonic fibroblasts as the feeder cell layer 10
• 8. Derivation and culture of putative pESC lines 10
• 9. Alkaline phosphatase activity detection and in vitro differentiation 11
• 10. Statistical analysis 11
• Ⅲ. RESULTS 13
• 1. Effect of novel system on blastocyst quality 13
• 2. Effect of novel system on attachment and outgrowth of blastocysts 20
• 3. Characterization of putative pESC lines 24
• Ⅳ. DISCUSSION 27
• Ⅴ. REFERENCES 30