ISOLATION AND CHARACTERIZATION OF PORCINE VISCERAL ENDODERM CELL LINES DERIVED FROM IN VIVO 11-DAY BLASTOCYSTS

Authors: Talbot, Neil; Blomberg, Le Ann; Mahmood, Ayesha; Caperna, Thomas; Garrett, Wesley

Submitted to: In Vitro Cellular and Developmental Biology - Animal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/12/2007
Publication Date: 3/16/2007
Citation: Talbot, N.C., Blomberg, L., Mahmood, A., Caperna, T.J., Garrett, W.M. 2007. Isolation and characterization of porcine visceral endoderm cell lines derived from in vivo 11-day blastocysts. In Vitro Cellular and Developmental Biology - Animals. 43(2):72-86.

Interpretive Summary: The work describes the isolation, development, and characterization of a porcine visceral endoderm cell lines (designated PE-1 and PE-2) that were derived from 11 day old ovoid or early tubular stage embryos, respectively. The endoderm is the second specialized tissue of the early embryo to form. Although the endoderm does not contribute to the embryo itself, i.e., is an extraembryonic tissue, it plays vitally important roles in development prior to when the placenta is formed. Most notably, visceral endoderm forms the ‘yolk sac’ which functions as a manufacturing site for early blood cells and blood proteins. Also, although the yolk sac does not literally surround a mass of yolk as in birds or reptiles, the tissue nonetheless still aides in providing nutrient flow from the mother’s uterus to the developing embryo. Pig embryos, particularly those made by in vitro fertilization techniques, have trouble surviving to the point that the placenta is formed and so often abort for that reason. The PE cell lines provides an in vitro model of porcine endoderm whose biological characteristics can be easily assayed and compared to other ungulate endoderm cell lines. Such comparative studies may lead to an understanding as to why porcine pregnancies may fail in early development. The cell lines may also be useful in efforts to improve the efficiency of animal cloning which is important because, via cloning, it may be possible to rapidly improve various traits of pigs.

Technical Abstract: Two porcine cell lines of yolk-sac visceral endoderm, designated PE-1 and PE-2, were derived from in vivo 11-day porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of cell lines was done on STO feeder cells. The PE-1 and PE-2 cells morphologically resembled visceral endoderm previously cultured from in vivo-derived ovine and equine blastocysts and from in vitro-derived bovine blastocysts. Analysis of the PE-1 and PE-2 conditioned medium by 2D-gel electrophoresis and MALDI-TOF mass spectroscopy demonstrated that they produced serum proteins. RT-PCR analysis showed the cells expressed several genes typical for yolk-sac endoderm or hepatocyte differentiation and function including GATA-6, DAB-2, REX-1, HNF-1, transthyretin, AFP, and albumin. However, unlike a porcine liver cell line, the PE-1 and PE-2 cell lines had relatively low inducible P-450 content and EROD activity, and, while they cleared ammonia from the cell culture medium, they did not produce urea. Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions in conjunction with lateral desmosomes. Rough endoplasmic reticulum was prominent within the cells. Immunocytochemistry indicated the PE-1 cells expressed cytokeratin 18 and had robust microtubule networks similar to those observed in in vivo porcine yolk sac endoderm. Metaphase spreads prepared at passage 26 of the PE-1 cell line indicated a diploid porcine karyotype of 38 chromosomes. The cells have been grown for over one year for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The cell lines will be of interest as an in vitro model of the porcine pre-implantation yolk sac.