Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines

Interest in producing lines of human embryonic stem cells is rapidly increasing because directed differentiation of these cells has the potential to provide treatments for numerous disorders, including diabetes, and to make large amounts of cells available for transplant treatments. This study assessed the use of donated gametes to establish human embryonic stem cell lines in the setting of an assisted reproductive technology program at an academic center. Oocytes were aspirated from 12 donors and inseminated with freeze-thawed sperm from two donors. All gamete donors were informed, verbally and in writing, that their oocytes and sperm would be used to produce embryos that in turn would not be used to initiate pregnancy. Oocyte donor cycles were suppressed with leuprolide acetate preceding human chorionic gonadotropin administration. Ovarian stimulation used recombinant FSH. Embryos were cultured to day 5 or 6 in sequential media. The inner cell masses of expanded blastocysts were isolated through immunosurgery, exposing the embryos to rabbit anti-BeWo antiserum and then guinea pig complement. The masses were plated onto primary mouse embryonic fibroblasts (PMEFs) irradiated to inactivate mitosis and cultured for 4 to 11 days. Viable cell colonies were passed onto fresh PMEFs at intervals of 7 to 10 days in the presence of leukemia inhibitory factor. Insemination of 162 mature human oocytes from the 12 donors yielded a 68% fertilization rate and a 50% rate of development to the expanded blastocyst stage. Blastocysts averaged 4.2 per oocyte donor. Immunosurgery on 40 blastocysts led to the culture of 18 inner cell masses that have produced three cell lines. All of these cell lines had the cellular and colony morphology described for human and rhesus monkey embryonic stem cells. One of the cell lines has stained positively for alkaline phosphatase and stage-specific embryonic antigen-4 (SSEA-4) and negatively for SSEA-1. It also expressed telomerase activity and produced human chorionic gonadotropin when allowed to differentiate. The investigators conclude, after internal and external reviews of the relevant ethical issues, that donated human gametes can be used to produce embryos for research.