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

Objective: To investigate the use of donated gametes for the production of human embryonic stem cell lines. Design: Basic research study. Setting: Assisted Reproductive Technology (ART) program at an academic inst itution. Patient(s): Consenting oocyte and sperm donors. Intervention(s): None. Main Outcome Measure(s): Oocytes were aspirated from oocyte donors (n = 12) and inseminated with frozen-thawed donor (n = 2) sperm followed by culture of embryos to day 5 or 6 in sequential media. The inner cell masses of exp anded blastocysts were isolated using immunosurgery and cultured for 4-11 d ays on irradiated primary mouse embryonic fibroblasts (PMEFs). Viable cell colonies were passed every 7-10 days onto fresh PMEFs in the presence of le ukemia inhibitory factor (0.1 mug/mL) and evaluated for appropriate cell su rface markers. Result(s): Immunosurgery of 40 blastocysts resulted in the culture of 18 in ner cell masses, which have produced three cell lines. One of these cell li nes has been shown to stain positive for alkaline phosphatase and stage-spe cific embryonic antigen (SSEA)-4 and negative for SSEA-1, express telomeras e activity, and produce hCG when allowed to differentiate. Conclusion(s): These findings demonstrate that the future production of hum an embryonic stem cell Lines for therapeutic use is possible with the use o f donated gametes. Many ethical issues were considered before the initiatio n of this study, and it was our goal to ensure that both oocyte and sperm d onors understood the nature and purpose of the research before their partic ipating in the study. (C) 2001 by American Society for Reproductive Medicin e.