Establishment Of Embryonic Stem Cells Through Improvement Of Porcine Parthenogenetic Blastocyst Quality

Abstract

Porcine parthenogenetic embryonic stem cells (ppESCs) have been successfully established but have not been maintained for large passage number. There are tremendous factors that affect the establishment of ppESCs, in which, blastocyst quality is one of them. Therefore, our main purpose of this study was to improve the blastocyst quality by adding the combination of fetal bovine serum (FBS), amino acid (AA, including 1% essential amino acid, 0.5% non-essential amino acid) and insulintransferrin-selenium (ITS) to the in vitro development (IVD) duration; and then use the high-quality blastocyst from the former experiment to improve the ppESCs establishment. In the first experiment, the oocytes were collected from 4-6mm in diameter of follicles and cultured to the morula stage. The morulae were then continuously cultured in 5 groups: FBS, FBS+ITS, FBS+AA, FBS+AA+ITS and control group on day 4 of IVD. In the next experiment, the inner cell mass (ICM) isolation from the high-quality blastocysts was performed by 2 methods: whole-blastocyst culture and ICM dissection method. The result showed that FBS+AA+ITS had the best performance with the significant high percentage in the hatched blastocyst day 8 (58.02%) and carrying significant high cell number. This result indicated that the combination of FBS+AA+ITS on day 4 had significant effect on the formation and hatching of blastocyst. With high quality, the hatched blastocysts day 8 from FBS+AA+ITS were kept on to the inner cell mass (ICM) isolation for ppESCs establishment. In the second experiment, the ICM dissection method had significant attachment rate, comparing to that of whole-blastocyst culture (44.98% vs 27.92%, p<0.05, respectively). Interestingly, the ICM colony from whole-blastocyst culture and ICM dissection method was able to be maintained over 5 passages with no significant differences (1.17% and 3.04%, respectively). This result suggested that both whole-blastocyst culture and ICM dissection did not effect on the ICM quality but only the attachment rate of ICM. In conclusion, the combination effect of FBS+AA+ITS significantly improved the quality and quantity of blastocyst. Additionally, the high quality of blastocyst together with the ICM dissection method were considered suitable for the establishment of ppESC. In the future, the porcine ICM colony should be continued to maintain for further culture to increase the passage number.