[The factors influencing the isolation of pig embryonic stem cells]

To study factors influencing the preparation of porcine ES cells, intact pig embryos at various stages of preimplantation development, as well as clusters of embryonic cells prepared from embryos at the stage of late or expanded blastocyst were cultivated in alpha-MEM, alpha-MEM + 50% BRL-conditioned medium, or in PBS + 20% FCS. NIH 3T3 cells, or pig granulosa cells were used as a feeder layer. It has been demonstrated that incubation conditions seriously affect development, release, and attachment to the feeder layer of both intact pig embryos and clusters of embryonic cells. However, the stage of preimplantation development is the most important factor, defining success in preparation of pig ES cells. It has been shown that embryoblast of pig embryos at stages of early, middle, late, and expanded blastocyst consists of 4-5, 8-10, 12-15, and 16-18 cells, respectively. This leads us to the conclusion that the rate of proliferation of ICM cells from pig embryos is very low. These results indirectly confirm the existence in embryonic development of pigs of metabolically resting ICM cells up to the beginning of gastrulation.