Isolation and characterization of porcine visceral endoderm cell lines derived from in vivo 11-day blastocysts.
Two porcine cell lines of yolk-sac visceral endoderm, designated as PE-1 and PE-2, were derived from in vivo 11-d porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of the 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 matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry demonstrated that they produced serum proteins. Reverse transcriptase polymerase chain reaction analysis showed that the cells expressed several genes typical for yolk-sac endoderm differentiation and function including GATA-6, DAB-2, REX-1, HNF-1, transthyretin, alpha-fetoprotein, and albumin. 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 and by lateral desmosomes. Rough endoplasmic reticulum was prominent within the cells. Immunocytochemistry indicated that 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 1 yr 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 preimplantation yolk-sac tissue.
Talbot
Neil C
NC
Biotechnology and Germplasm Laboratory, Animal and Natural Resources Institute, ARS, USDA, Bldg. 200, Rm. 13, BARC-East, Beltsville, MD 20705, USA. Ntalbot@anri.barc.usda.gov
Blomberg
Le Ann
LA
Mahmood
Ayesha
A
Caperna
Thomas J
TJ
Garrett
Wesley M
WM
D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D001798:Blood Proteins / Q000096:biosynthesis; D002460:Cell Line; D048430:Cell Shape; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins / Q000096:biosynthesis; D004707:Endoderm / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D015870:Gene Expression; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D013552:Swine; D015017:Yolk Sac
D001798:Blood Proteins; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins