Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig.

Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.

Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer.

Cryopreservation could be a useful technique for providing a steady source of oocytes for nuclear transfer and in vitro embryo production. The purpose of this study was to develop a method for cryopreservation of bovine oocytes while maintaining the developmental potential following subsequent in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Following vitrification-thawing, the surviving oocytes were (a) used for parthenogenetic activation, (b) examined for pronuclear formation after IVF, (c) examined for embryo development after IVF, and (d) used for SCNT employing fetal fibroblasts transfected with green fluorescent protein (GFP) gene. While most of the oocytes survived vitrification when the microdrop method was used (92.50%), the cleavage and blastocyst formation rates after parthenogenetic activation were lower (46.5% and 11.1%) than that in the non-vitrified control (86.6% and 13.5%). After IVF, the pronuclear formation (2PN) of fertilized embryos was lower in the vitrified group than in the control (21.7% and 59.9%). After SCNT, fusion rates were similar in control (58.33%) and vitrified-thawed oocytes (53.19%). However, the cleavage (73.1% and 46.3%) and blastocyst formation rates (22.2%, 7.4%; p<0.05) differed between control and vitrified-thawed oocytes. In vitrified-thawed or control oocytes, all embryos reconstructed using fetal fibroblasts transfected with GFP gene showed GFP expression. To evaluate the complete developmental potential, embryos derived from vitrified-thawed and fresh control oocytes were non-surgically transferred to 27 recipients (16 for control and 11 for vitrified-thawed). In the vitrified-thawed group, two pregnancies were detected at day 60, and one of them lasted until day 222. While in the fresh group, one pregnancy maintained to term. In conclusion, vitrified-thawed bovine oocytes could support development into the subsequent stages after IVF and SCNT. In addition, this study showed the possibility of the vitrified-thawed bovine oocytes in the production of transgenic cloned animals. In addition, further studies are required to increase the efficiency of oocyte vitrification for the practical uses and production of live offspring.

In vitro development and cell allocation of porcine blastocysts derived by aggregation of in vitro fertilized embryos.

In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to those of their in vivo counterparts. The objective of this study was to increase developmental competence and to gain an understanding of cell allocation in blastocysts derived from the aggregation of four-cell stage porcine embryos produced in vitro. After removal of the zona pellucida, two (2x) and three (3x) four-cell stage embryos were aggregated by co-culturing them in aggregation plates. Five days after aggregation, the developmental ability and the number of cells in the aggregated embryos were determined. The percentage of blastocysts was higher (P < 0.05) in both the 2x and 3x aggregated embryos (66.6% and 72.0%, respectively) compared to that of the 1x embryos and the intact controls (43.1% and 36.4%, respectively). The total cell number of blastocysts also increased in aggregated embryos compared to that of intact controls (2.6-fold for 2x and 3.4-fold for 3x) (P < 0.05). The cells of two differentially stained embryos were started to mix at 72 hr after aggregation. In vitro-fertilized porcine aggregates (2x) were developed to blastocyst with a random distribution of cells from each embryo. The mRNA levels for the oct-4, bcl-xL and connexin 43 genes were higher (P < 0.05) and bak gene were lower (P < 0.05) in both the 2x and 3x aggregated embryos than the intact controls. Therefore, the aggregation of the four-cell stage embryos could be used to improve the quality of porcine preimplantation stage embryos produced in vitro.