279 COMPARISON OF DIFFERENT METHODS OF ISOLATION OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO-PRODUCED GOAT BLASTOCYSTS

Abstract

The present study was carried out to compare different isolation procedures for embryonic stem (ES) cell-like cells from in vitro-produced goat blastocysts. Goat oocytes were aspirated from slaughterhouse-derived ovaries; matured in vitro in maturation medium containing TCM-199 (HEPES modified) with 5 µg mL–1 FSH, 10 µg mL–1 LH, 1 µg mL–1 estradiol-17β, BSA, and 10% estrous goat serum; and fertilized by freshly collected buck semen. The fertilized oocytes were cultured in embryo development medium containing TCM-199 (HEPES modified), 50 µg mL–1 sodium pyruvate, 3.5 µg mL–1 L-glutamine, gentamicin, essential amino acids, nonessential amino acids, BSA, and 10% fetal calf serum, along with goat oviductal cells for further development. A total of 250 blastocysts and 50 hatched blastocysts were used to isolate and culture goat ES cell-like cells. Inner cell mass cells (ICMs) were isolated mechanically from 100 blastocysts and 20 hatched blastocysts and enzymatically from 50 blastocysts and 15 hatched blastocysts. The ICMs were cultured on 10 µg mL–1 mitomycin-C-inactivated goat fetal fibroblast feeder layer. Primary colony formation was significantly higher (P < 0.01) when hatched blastocysts were used for ICM isolation (28/35; 80%) than when blastocysts were used (77/150; 51.3%). However, when ICMs were isolated mechanically, the percentage of primary colony formation was significantly higher (P < 0.01) for blastocysts (66/100; 66%) as well as for hatched blastocysts (18/20; 90%) than when ICMs were isolated enzymatically (15/50; 30%, and 11/15; 73%), respectively, for blastocysts and hatched blastocysts. ES cell-like cells were also isolated from intact blastocysts and intact hatched blastocysts. But the primary colony formation (27/100; 27%), and (6/15; 40%), respectively, was significantly low (P < 0.01) compared to that for mechanically isolated ICMs. Statistical analysis was done by chi-squire test. When the putative ES cell-like cells were subcultured enzymatically (0.25% trypsin–EDTA), they differentiated after 3 passages. However, when the mechanical subculture procedure was followed, the ES cell-like cells maintained their pluripotent state up to the 9th passage, and three goat ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). The putative ES cell-like cells were stained with alkaline phosphatase substrate solution (Sigma, St. Louis, MO, USA). The RT-PCR mediated expression of Oct-4 was done by a special kit Cells-to-cDNA™ II (Ambion, Austin, TX, USA). The ES cell-like cells expressed both alkaline phosphatase and Oct-4. It may be concluded that mechanical isolation of hatched goat blastocysts is the best method for isolation of goat embryonic stem cell-like cells, and they are positive for alkaline phosphatase and Oct-4.