CULTURING THE EPIBLAST CELLS OF THE PIG BLASTOCYST

Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was use d to isolate the epiblast from trophectoderm and primitive endoderm be fore culturing. Blastocysts collected at 7 to 8 days postestrus were i mmunodissected to obtain the inner cell mass (ICM) and destroy trophec todermal cells. The ICM was cultured for 2 to 3 days on STO feeder cel ls. The epiblast was then physically dissected free of associated prim itive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This p rimary cell morphology changed as the colonies grew and evolved into t hree distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously dif ferentiated into numerous specialized cell types in STO co-culture. Th ese included fibroblasts, endodermlike cells, neuronlike cells, pigmen ted cells, adipogenic cells, contracting muscle cells, dome-forming ep ithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithel ium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an ind efinite life span in STO co-culture and also retained a normal karyoty pe. These results demonstrate the in vitro pluripotency of pig epiblas t cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.