383 IDENTIFICATION OF PLURIPOTENCY MARKERS IN SWINE EMBRYOS

Abstract

Embryonic stem cells (ESC) are a useful tool for studying embryonic development, cell differentiation, and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral, and religious considerations by scientists and laymen alike. Therefore, an animal model such as the pig (Sus scrofa) is valuable in overcoming such hurdles because this species holds physiologic parameters similar to humans. In spite of the great biomedical potential of ESC, many difficulties have been faced in maintaining these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro- and in vivo-produced swine blastocysts; (2) to compare 2 in vitro culture conditions to maintain isolated inner cell masses (ICM), murine embryonic fibroblasts (MEF), or Matrigel; and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2, and FoxD3 at ESC and in vitro- and in vivo-produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vivo-produced blastocysts were obtained by superovulation followed by AI of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing 5 days after ovulation. In vitro- and in vivo-produced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution, and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. Resultant ICM (14 and 66 ICM from in vitro- and in vivo-produced blastocysts, respectively) were cultured in stem cells media (GMEM added by 15% FCS, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 4 ng mL^-1 of basic fibroblast growth factor) over monolayer of irradiated mouse embryonic fibroblasts (MEF) or Matrigel for 3 weeks. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro- and in vivo-produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis was performed only with whole in vitro- and in vivo-produced blastocysts. Results showed that Nanog and Sox2 were less expressed in in vitro-produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Because no ESC lineage was obtained in swine until now, we believe this species has different requirements compared with murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC.

Acknowledgments are given to FAPESP (processes 06/58507-0 and 07/51732-0).