Cheong Seung A, Kim Eunhye, Kwak Seong-Sung, Jeon Yubyeol, Hyun Sang-Hwan
Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
Mol Med Rep. 2015 Aug;12(2):2140-8. doi: 10.3892/mmr.2015.3634. Epub 2015 Apr 16.
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.
猪胚胎干细胞（pESCs）在转化生物医学研究中具有巨大的应用潜力，包括异种移植和疾病模型。获得高质量的囊胚是原代胚胎干细胞分离、定植及建立胚胎干细胞系的最重要因素。在猪中，与体内来源的囊胚相比，体外培养获得的囊胚细胞数量有限，且内细胞团不确定，这可能导致无法建立猪胚胎干细胞系。在本研究中，研究了白藜芦醇（RES）、粒细胞巨噬细胞集落刺激因子（GM-CSF）和β-巯基乙醇（β-ME）对囊胚质量和集落衍生效率的影响，以建立胚胎干细胞系。开发了一种新的培养系统，即在卵母细胞体外成熟（IVM）培养基中添加2µM RES，在胚胎体外培养（IVC）培养基中添加10 ng/ml猪GM-CSF和10µMβ-ME。与对照系统相比，该新系统在第7天显示出显著更多的孤雌激活（PA）囊胚（54.5±1.8%对43.4±1.2%；P<0.05）和体外受精（IVF）囊胚（36.9±3.3%对26.2±2.9%；P<0.06）。与对照系统相比，新系统的PA和IVF囊胚在第7天显示出显著更高的孵化率（P<0.05）和更多的细胞数量（PA为55.1±2.0对45.6±2.0；P<0.05，IVF为78.9±6.8对58.5±7.2；P<0.06）。接种到饲养细胞上后，新系统产生的PA囊胚显示出显著提高的附着率（28.8±3.9%对17.2±2.4%；P<0.062）。最后，建立了来自新系统产生的PA胚胎的两个假定的pESC系和来自对照系统的一个假定的pESC系。总之，新系统提高了囊胚质量，并提高了体外产生的猪PA和IVF胚胎的假定pESC系的衍生效率。
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