Generation of chimeric piglets by injection of embryonic germ cells from inbred Wuzhishan miniature pigs into blastocysts.

ABSTRACT

BACKGROUND: Human embryonic stem/germ (ES/EG) cell research poses ethical dilemma, it is therefore critical to establish alternative sources of cells for relevant studies. Considering the similarities between the inbred miniature Wuzhishan pigs (WZSP) and humans, ES/EG from these pigs can serve as potential substitutes in human research. In this study, we reported our results that successfully established stable EG cell lines from the WZSP. METHODS: Primordial germ cells (PGCs) were isolated from the genital ridges of pig fetuses at 25 to 28 days of pregnancy. To obtain stable EG cell line, PGCs were maintained on STO cells in DMEM containing multiple essential growth factors. RESULTS: Two EG cell lines were established and characterized by positive alkaline phosphatase staining (AKP), expressions of Oct-4, SSEA-1, SSEA-3, SSEA-4, ability to differentiate into cells of all three germ layers in vitro, and generation of chimeric offsprings after microinjection and embryo transfer. Transmission electron microscopy demonstrated that the cytoplasmic structure of pig EG cells were rather simple and had a higher nuclear-to-cytoplasm ratio. Scanning electron microscopy showed the sizes of pig EG cells were similar to mouse EG cells. Both EG cell lines showed normal karyotypes. The EG cells were propagated for more than 20 passages and underwent multiple cycles of freezing and thawing, without losing their pluripotency (as distinguished by AKP staining). CONCLUSIONS: Both in vitro and in vivo evidence strongly demonstrated that EG cells harvested from the inbred miniature WZSP were pluripotent and can be used for relevant pig or human studies.