The Generation Of Embryonic Stem Cell Line From Mâ…¡ Oocyte Derived From Ovarian Grifts Of Newborn Mouse

Object:1.To establish a tissue engineering technology model for harvestingdevelopmentally competent MⅡ oocytes from primordial follicles.2. To generateembryonic stem cell line using artificial-developed MⅡ oocytes derived fromprimordial follicles.3.To build an experimental basis for human ovarian project.Method: The fresh half-ovarian grafts from the newborn mice were immediatelytransplanted to kidney capsule of recipient mice to initiate oocyte growth. Grafts wererecovered at23days after received10IU PMSG for44h. Subsequently, the COCsharvested from graft were cultured for16h in vitro to generate mature oocytes.Embryonic stem cell–like colonies were established by subculturing the inner cellmass cells of blastocysts developed from in vitro fertilized graft-oocytes.Result(s):1. One colony-forming cell line was established by IVM-MⅡoocytesobtained from COCs of grafted ovary slices.2. One established stem cells line havesuccessfully subcultured for4passages（P4）.3. Colony-forming cells at the3thsubpassage stained positive for alkaline phosphatase(AP), anti-SSEA-1. Thecolony-forming cells were transformed into a teratoma consisting of three germlayer-derived cells after being transplanted into kidney capsule of male mice.Conclusion(s):1. To our knowledge, this is the first time that ESC have beenproduced by oocytes developed from non-recruitment primordial follicles of newbornmice’s ovary slices.