This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Through responsible embryonic stem cell research in closely related species such chimpanzees, we hope to work towards cellular based therapies for many disorders including diabetes, Alzheimer's, Parkinson's, and cerebral palsy just to name a few. The overall goal of this pilot study is to derive and fully characterize stable cultures of chimpanzee embryonic stem cells (cESC). In order to do this, female chimps will be visually monitored in order to determine the stage of their menstrual cycle. Once at the proper stage, the female will be hormonally stimulated with a combination of follicle stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG). This hormonal stimulation regimen increases the number of mature oocytes that will be produced during the animal's menstrual cycle. Once stimulated, oocytes will be harvested using a technique known as trans-vaginal, ultrasound guided oocyte aspiration or TVA. This non-invasive procedure utilizes a special ultrasound probe that is placed into the vaginal vault of the animal and allows for the visualization of ovarian follicles. Once follicles are visualized, a needle is extended from the ultrasound probe, through the vaginal wall and into the follicle. Suction is applied and the contents of the follicle, including the oocyte, are collected into a sterile tube. The procedure is repeated until all follicles are harvested. After all follicles are harvested, the collected aspirates will be taken to the laboratory, where they will be cleaned and searched for the oocytes. The oocytes will be isolated into a culture dish and separated based on their quality and maturity. On average, in rhesus and baboons, we obtain approximately 30 oocytes per stimulation of which 60% are typically mature/usable. Once the oocytes reach full maturity, they will be fertilized using chimp sperm collected the same day via rectal probe electroejaculation. After fertilization, the embryos will be cultured in the laboratory until they reach the blastocyst stage. At this point, immunosurgery will be performed on each embryo in order to isolate the inner cell mass. The isolated inner cell mass will then be cultured on a layer of feeder cells where it will, hopefully, continue its growth and form a stable colony of undifferentiated embryonic stem cells.
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