Coaxing HESC to form Oocyte-Like Structures by Co-Culture with Testicular Extract and Hormones

Reports have shown that murine embryonic stem cells (mESC) can differentiate into primordial germ cells (PGC) and then to early gametes (oocytes and sperm) that after fertilization form blastocysts. Preliminary studies indicated that hESC also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of hESCs into oocytes. Here, we investigated the effects of human fetal testicular extracts (HFTE) and hormones i.e. follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG) to coax hESC to differentiate into oocytes. The embryoid bodies (EBs) derived from hESC formed ovarian-like structures (OLS) after treatment with HFTE for 7 days and with hormones (FSH + hCG) for 20-30 days. OLS exhibited typical oocyte-like spherical shape of variable sizes and some with zona pellucida-like covering RT-PCR, immunological fluorescence staining and flow cytometry analyses showed that these structures are positive for specific germ cell and oocyte markers such as OCT4, SCP3, C-KIT and DDX4, STELLA, FIGLA, GDF9, NANOG,FSHR, ZP1, ZP2 and ZP3. Quantitative RT-PCR analysis showed that FSHR, GDF9 and FIGLA were up-regulated during OLS differentiation from EB in a time-dependent manner. These results demonstrated that hESC have the ability to differentiate into ovarian structures. This study thus provides an in vitro model to study germ-cell formation from hESC and these germ cells may generate a potential source of oocytes for therapeutic cloning.