194 ESTABLISHMENT OF GOAT EMBRYONIC STEM CELL-LIKE LINES DERIVED FROM IN VIVO-PRODUCED BLASTOCYST-STAGE EMBRYOS

Abstract

Pluripotent embryonic stem cells (ESC) derived from the inner cell mass (ICM) of mammalian blastocysts provide AN unlimited number of cells that can be used in gene targeting and be of great value to agriculture and medicine. Embryonic stem cells with capacity for germ line transmission have been verified only in mouse despite many efforts to derive ESC from other mammalian species. The methods for the derivation, propagation and differentiation of ESC from domestic animals have not been fully established. The objective of this study was the generation and initial characterization of goat embryonic stem cells (GESC) derived from in vivo-produced blastocyst-stage embryos. Goat compact morulae and blastocysts were collected from superovulated adult Saanen crossbreed donors 7 days after insemination with a fertile male. Embryos were collected by uterine flushing using a 12Fr. Foley catheter by means of a laparoscopically assisted mid-ventral laparotomy under general anesthesia. Twenty eight in vivo-derived blastosyst-stage embryos were cultured on a goat fetal fibroblast feeder layer (inactivated by Mitomycin C) in a medium of DMEM containing 0.1 mm ²-mercaptoethanol, 0.1 mm MEM nonessential amino acids, 200 mm l-glutamine, and 10% FCS. Following three days in culture 25 of 28 embryos hatched. Ten embryos attached to the feeder layer, nine degenerated, and nine embryos were floating in the medium and expanding in size. After 5-7 days in culture four of the tehn attached embryos appeared with a prominent an ICM outgrowth and 3 of the nine floating embryos formed structures resembling ICM disc surrounded by trophectoderm cells. The ICM and the embryonic discs were isolated mechanically and cultured on goat feeder cells in DMEM medium containing 10 ng/mL horse leukemia inhibitory factor (hLIF) and 10% FCS. The ICM and embryonic disc outgrew into colonies on Day 4 post-culture. Compact colonies of cells from these outgrowths were isolated mechanically and passed onto fresh goat feeder cells every 4-5 days with the addition of 10 ng/mL hLIF to the culture media. Established colonies at passage 6 were tested for immunoreactivity against alkaline phosphatase (AP) and Oct-1 using standard protocols. The result was the estabilshment of embryonic stem cell-like colonies (57%) from both ICM and embryonic discs cultured on goat fetal fibroblast feeder cells. Colonies forming from these outgrowths (50%) of either ICM or embryonic disc stained positive for both AP and Oct-4. Embryoid bodies formed from colonies of either ICM or embryonic disc in suspension (DMEM containing 10% FCS with no hLIF). Two cell lines (one from ICM and one from embryonic disc) have been maintained so far through passage 8 and have been cryopreserved. These GESC-like lines will be used in further characterization and ultimately in transgenic animal production studies.