Establishment of embryonic stem cell lines from the dog and their application to regenerative veterinary medicine

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Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. The objective of this study was to establish ES cell lines from the dog for regenerative veterinary medicine, and the following results were obtained.1.The rise in the levels of hypothalamic P450 aromatase mRNA, hypothalamic and pituitary estrogen receptor α and β mRNAs, and plasma estradiol-17β was associated with termination of anestrus in the bitch.2.The intranasal administration of a depot-injectable form of the potent GnRH agonist leuprolide acetate was a useful method for controlling the induction of estrus in anestrous bitches that appears to cause the animals little stress, and a combination of leuprolide acetate and oral estradiol-17β might be more effective in inducing estrus.3.Although the in vitro maturation rate of canine MI oocytes cultured with estrogen and epidermal growth factor was higher than for oocytes cultured in culture media only, the rate that reached the MII stage w … More as not different in both groups.4.Co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes, and co-culture with canine embryonic fibroblasts developed the embryo up to the 16-cell stage, and with mouse embryonic fibroblasts up to morula stage.5.Primary canine ES-like cell colonies were formed in 67.9% of inner cell masses from hatched blastocysts, which was significantly higher than from blastocysts (25.6%) and d morulae stage (0%).6.Canine ES-like cells had typical stem cell-like morphology, expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4, and formed embryoid bodies (EBs) in a suspension culture ; extended culture of EBs resulted in the formation of cystic EBs. When the simple Ms were cultured in tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like and myocardium-like cells.7.Dissociation of canine ES-like cell colonies with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remain undifferentiated, and two ES-like cell lines passed 8 passages. Less