The overall objective of this protocol is to isolate and characterize baboon embryonic stem (ES) cells for the purpose of providing an in vitro model of early cell growth and proliferation similar to that in humans, and as a source of differentiation-driven cells, tissues, and organs. The baboon is used as a model of bone marrow transplantation, and an ES cell line will be a useful source of blood cell precursors, thus helping to conserve the number of animals for biomedical research. Five adult female baboons were observed on a daily basis and paired with adult male baboons at the onset of sexual receptivity, which was determined by change in the color and swelling of the perineal sex skin. Observations of mating behavior were recorded and the pair was separated when the female was no longer receptive (around the time of detumescence or decreasing color and swelling of the sex skin). Based on previous studies in the baboon, ovulation was estimated to occur three days before the onset of detumescence. Nonsurgical attempts to recover preimplantation embryos were made five to nine days after ovulation. Baboons were sedated with 0.2 ml/kg of ketamine/xylazine (0.70.3) and placed in lateral recumbency. A 14-gauge cannula (stainless steel tube) was passed through the vagina and cervix and into the uterus. Ultrasound was performed to confirm uterine placement. Sterile culture medium was flushed into the uterus and the back flow was collected into a sterile culture tube. A microscopic search for the embryo was made. Any embryos found were shipped via express mail to the Wisconsin Regional Primate Research Center for stem cell derivation. Over the past ten months, 12 flush attempts were made. Nine of these were attempted on animals that mated successfully. Four of these nine animals were flushed twice in one month, but the second flush also did not result in any embryos. One embryo was collected and sent to Wisconsin. It arrived safely and continued to do well in culture. Continued efforts will help assure the successful establishment of baboon ES cells as an important and useful in vitro cell culture system.
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