281 ATTEMPTS FOR ESTABLISHMENT OF PORCINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

Abstract

The porcine embryonic stem (ES) cells could be a useful tool for the production of transgenic animals and the study of developmental gene regulation. Even though the efficiency of establishment of ES cells from in vivo blastocysts is relatively high, especially in mice, it is difficult and expensive to obtain in vivo embryos in domestic animals. Recent development of techniques in the production of embryos in vitro could be a useful source for the establishment of ES cells. However, the morphology and cell quality of in vitro-produced embryos are inferior to those of their in vivo counterparts. Although many attempts have been made to establish ES cells from in vitro-produced embryos, the overall efficiency is extremely low because of the poor embryo quality. However, aggregation of in vitro-produced embryos was developed to increase the number of cells in the inner cell mass (ICM) of blastocysts and could be useful in the application to ES cell establishment. Therefore, in this study, we attempted to derive porcine ES cells by using aggregation of in vitro-produced embryos by in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Embryos at the 4-cell stage were produced by culturing embryos for 2 days after IVF and SCNT. After removal of the zona pellucida with acid Tyrode’s solution, three 4-cell-stage embryos (IVF3X) from IVF and two 4-cell-stage embryos (NT2X) from SCNT were aggregated by co-culturing them in an aggregation plate followed by culturing to the blastocyst stage. Embryos from IVF (IVF control) and SCNT (NT control) were also cultured to the blastocyst stage. All blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Two primary colonies were formed in the IVF control group (3.9%), whereas four primary colonies were formed in the IVF3X group (12.5%). One primary colony was formed in the NT2X group (20%), although no colony was formed in the NT control group. One of the IVF3X lines gradually disappeared after sub-passing, and the NT2X line also disappeared. Two ES-like cell lines derived from the IVF control were maintained up to 14 passages, and three ES-like lines from IVF3X were also maintained for more than 14 passages. These cells morphologically resembled human ES cells (flat and single layered) and expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, Oct-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. These results indicated that a porcine ES cell line could be established from in vitro-produced aggregated blastocysts. Further research is required to establish ES cell lines from SCNT embryos and characterize the differentiation and developmental abilities of these porcine ES-like cells. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).