Isolation of female germline stem cells from porcine ovaries and differentiation into oocytes like cells

Abstract

The establishment of female germline stem cells (FGSCs) and their differentiation ability into oocyte – like cells (OLCs) in large mammals is controversial because of questions regarding the potential to produce offspring and the mechanism of FGSCs self-renewal remains unclear. Thus, in order to study more about this field, there needs to figure out an ideal medium with purpose that supporting for successfully isolation of FGSCs and differentiation into OLCs. We used the culture medium containing DMEM/F12 supplied with 2% N21 to isolate FGSCs from porcine ovaries. The isolated FGSCs were characterized with germ cell marker VASA and OCT4 and continued to examine the differentiation potential into oocyte-like cells by culturing on a MEF monolayer as feeder layer and co – culture with MEF cells. The obtained OLCs were characterized with oocytes markers VASA, GDF9 and epigenetic marker AcH3K9. FGSCs were successfully isolated and cultured for 1 week with medium containing DMEM/F12 supplied with 2% N21 and strongly expressed germ cell marker VASA and OCT4. Under two differentiated conditions, the obtained FGSCs spontaneously differentiated into oocyte-like cells in vitro. However, the OLCs which were formed in co –culture systems were extremely larger in number and size in comparison to the ones were cultured on a feeder layer. Moreover, the obtained OLCs were characterized by immunostaining and strongly expressed the oocyte markers GDF9, VASA and transcription marker AcH3K9. Our results proved that under optimal condition in co –culture with MEF in 3D low attachment dish, FGSCs could differentiate into OLCs at 37oC and 5% CO2. Furthermore, these OLCs had the ability to enlarge up to the size similar to oocytes and strongly express oocyte markers. Keywords: Female germline stem cells, Oocytes – like cells, differentiation, proliferation, immunostaining