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.

Carboxyethylgermanium sesquioxide (Ge-132) treatment during <i>in vitro</i> culture protects fertilized porcine embryos against oxidative stress induced apoptosis

“…Also, the derivation efficiency of ESC might be another tool to evaluate blastocysts indirectly [ 56 ]. Some reports have shown that reducing oxidant stress increased the success of establishing new mouse and porcine ESC lines [ 57 , 58 ]. Although further experiments need to be conducted, the putative porcine ESC lines were derived from 100- and 200-µg/ml Ge-132 treatment group and showed a similar derivation process with primed ESCs, such as previously reported porcine ESC lines [ 59 ] as shown in Supplementary Table 1 and Supplementary Fig.…”

Resveratrol enhances pluripotency of mouse embryonic stem cells by activating AMPK/Ulk1 pathway

“…The beneficial effects of resveratrol on a large number of cellular processes allowed us to assume that this promising compound can also be useful in the positive regulation of the fundamental properties of ESCs—self-renewal and pluripotency. In favor of this assumption, there is an evidence that supplementation of resveratrol has beneficial effect on porcine and cow in vitro fertilization and subsequent embryonic development 11,12 . The addition of resveratrol to the medium for cultivation of pig oocytes allows to obtain more viable blastocysts and efficiently isolate ESCs from them 11 .…”

Supplementation of kaempferol toin vitromaturation medium regulates oxidative stress and enhances subsequent embryonic developmentin vitro

“…Improvements to the IVM system will benefit from a range of applications, including animal husbandry and medicine, in addition to the potential use of planned in vitro fertilization, including efficient embryo production, cloning, genetic modification and animal models (de Souza-Fabjan et al, 2014). Compared with other animals, porcine animals have become the model of choice for human transformation studies due to their similarity in body shape and physiological structure (Cheong et al, 2015). Although in vivo embryos are better than in vitro embryos, it is more expensive and laborious to produce embryos in vivo.…”