Abstract

Embryonic stem cells (ESC) provide potentially unlimited cell numbers for gene targeting, providing great value to agriculture and medicine. This study was conducted to isolate and characterize parthenogenetic ESC (pESC)-like cells from in vitroproduced buffalo parthenotes. A total of 100 blastocysts were produced through chemical activation. For pESC culture, 20 blastocysts were left intact, while the rest was dissociated mechanically or enzymatically, to isolate inner cell mass (ICM) cells from 44 hatched and 36 non-hatched blastocysts, respectively. ICM cells were seeded and cultured on mitomycin-C treated feeder layers of buffalo fetal fibroblasts. Primary cell colonies formed three tofour days after ICM seeding. Colony formation was significantly lower from intact blastocysts (3/20 (15%)) than from ICM cultures (39/80 (49%)) with hatched blastocysts being better starting material than non-hatched ones (30/44 (68%) versus 9/36 (25%), respectively). Up to passage nine, two mechanically isolated pESC-like lines remained viable and expressed specific markers such as alkaline phosphatase activity, as well as Nanog and Oct-4 mRNA. Cells formed embryoid bodies in suspension culture. It has been shown it is possible to isolate buffalo pESC-like cells from parthenogenetically developed embryos and maintain the culture in vitro for a prolonged period of time.

V, Verma, and MS Chauhan

Proceedings of the New Zealand Society of Animal Production, Volume 68, Brisbane, Australia, 4-7, 2008