192isolation and Culture of Embryonic Stem Cell-Like Cells From in Vitro Fertilized Porcine Blastocysts

Abstract

The establishment of porcine embryonic stem (ES) cell lines should be useful for the production of transgenic pigs and studies of developmental gene regulation. Recent development of techniques for production of embryos in vitro could be a useful source for the isolation of ES cells. Therefore, to establish porcine ES cells, this study was conducted to isolate and culture inner cell mass (ICM) from in vitro-fertilized (IVF) porcine blastocysts. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Oocytes were then fertilized using a modified swim-up method to prevent polyspermy and cultured to the blastocyst stage. Initial culture of ICM was conducted after either culture of whole embryos or isolation of ICM by immunosurgery. Developing IVF embryos were continuously cultured in 50% DMEM and 50% F-10 with 15% fetal bovine serum, 1% non-essential amino acids, 1.7mM L-glutamine, 1% penicillin/streptomycin, 0.1mM α-mercaptoethanol, 1000 unit recombinant human LIF, 40ngmL−1 recombinant human SCF and 20ngmL−1 recombinant human basic FGF on a mytomycin-C-inactivated murine embryonic fibroblast (MEF) feeder layer. Antibodies against porcine cells were produced in rabbit. After removal of zona pellucida, ICMs were isolated by immunosurgery and cultured on feeder cells the same as described above. After IVF, the rates of 2-cell embryos and blastocysts were 70.8% and 20.4%, respectively. Results from the isolation and culture of ICMs of porcine blastocysts are shown in following table. ICM isolated by immunosurgery showed better attachment to feeder cells and ES cell colony formation than cultured whole blastocysts. Morphology of colonies was similar to that of mouse ES cells, showing compact colonies with delineated boundary. Also, these colonies showed alkaline phosphatase activity. Porcine ES-cell like colonies were passed 3 times through physical separation on fresh feeder layers. These results indicated that porcine ES-like cell line can be established from IVF porcine blastocysts. Further characterization of these porcine ES-like cell lines is required. Table 1 Isolation and culture of ICM from porcine blastocyst produced by IVF