Lanzendorf S E, Boyd C A, Wright D L, Muasher S, Oehninger S, Hodgen G D
Technology Development Center, Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.
Fertil Steril. 2001 Jul;76(1):132-7. doi: 10.1016/s0015-0282(01)01825-8.
To investigate the use of donated gametes for the production of human embryonic stem cell lines.
Basic research study.
Assisted Reproductive Technology (ART) program at an academic institution.
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 expanded blastocysts were isolated using immunosurgery and cultured for 4-11 days on irradiated primary mouse embryonic fibroblasts (PMEFs). Viable cell colonies were passed every 7-10 days onto fresh PMEFs in the presence of leukemia inhibitory factor (0.1 microg/mL) and evaluated for appropriate cell surface markers.
RESULT(S): Immunosurgery of 40 blastocysts resulted in the culture of 18 inner cell masses, which have produced three cell lines. One of these cell lines has been shown to stain positive for alkaline phosphatase and stage-specific embryonic antigen (SSEA)-4 and negative for SSEA-1, express telomerase activity, and produce hCG when allowed to differentiate.
CONCLUSION(S): These findings demonstrate that the future production of human embryonic stem cell lines for therapeutic use is possible with the use of donated gametes. Many ethical issues were considered before the initiation of this study, and it was our goal to ensure that both oocyte and sperm donors understood the nature and purpose of the research before their participating in the study.
研究使用捐赠配子来生产人类胚胎干细胞系。
基础研究。
一所学术机构的辅助生殖技术（ART）项目。
同意参与的卵母细胞和精子捐赠者。
无。
从卵母细胞捐赠者（n = 12）处吸出卵母细胞，用冻融的捐赠者（n = 2）精子进行授精，然后在序贯培养基中将胚胎培养至第5天或第6天。使用免疫外科方法分离扩张囊胚的内细胞团，并在经辐照的原代小鼠胚胎成纤维细胞（PMEF）上培养4 - 至11天。每7 - 10天将存活的细胞集落传代到含有白血病抑制因子（0.1μg/mL）的新鲜PMEF上，并评估其是否具有合适的细胞表面标志物。
对40个囊胚进行免疫外科操作后培养出18个内细胞团，从中产生了3个细胞系。其中一个细胞系已被证明碱性磷酸酶和阶段特异性胚胎抗原（SSEA）-4染色呈阳性，SSEA-1染色呈阴性，具有端粒酶活性，在诱导分化时可产生hCG。
这些发现表明，使用捐赠配子未来有可能生产用于治疗的人类胚胎干细胞系。在开展本研究之前考虑了许多伦理问题，我们的目标是确保卵母细胞和精子捐赠者在参与研究之前了解研究的性质和目的。
