Differentiation Potential Of Mouse Escs Into Germ Cells And Their Development In Vivo

Embryonic stem cells (ESCs) are undifferentiated cells that derived from early preimplantation blastocyst inner cell mass or primordial germ cells, which have the potential of self-renewal, highly proliferation and differentiation. Embryonic stem cells both in vitro and in vivo have the ability to differentiate into all three germ layers, which has been widely proven. The occurrence of germ line cells has already been a hot topic in life science research. Compared to somatic cells, germ cells have unique biological features. However, in mammals, the precise time and position of germ-line cell specialization as well as the intrinsic biological property is still an issue in developmental biology.In recent years, some research related to embryonic stem cell differentiation to germ cell has been reported. However, the induction efficiency is still low. How to increase the induction efficiency and impove the induction method have turned to be a concerned problem. In this study, firstly, based on the mouse embryonic stem cells with the report plasmid under the promoter of Figlα(Figlα-ESCs) constructed by our group, oocyte extracts and co-culture with granulosa cells were applied to induce embryonic stem cells to differentiate to female germ cells, trying to establish a model for the research on reproductive female germ cells in vitro. Then using the model of the germ cell-deficient mice caused by busulfan as a platform, transplant the Figlα-ESCs in vivo, and discuss the differentiation potential of mouse embryonic stem cells in mouse body microenvironment.1. Extracts from the materials of fish ovary oocytes were prepared and Figlα-ESCs were induced to differentiate with the extracts above three different concentrations, 10 mg/mL, 20 mg/mL, and 50mg/mL, respectively. The immunofluorescence and Fluorescence quantitative PCR of 3 d and 7 d of induction shows that when adding extracts without feeder conditions in the short-term, ES cells can maintain its undifferentiated form. After prolonging the induction time, some cells treated by the extracts growed big and round and expressed some germ cell and ovary oocyte specific genes, such as VASA, SCP3 etc. 2. Induced the fusion of Figlα-ESCs by fish ovary oocyte extracts, after 7 days there being no obvious morphological changes observed. Preliminary results showed that the fusion induction did not impact the differentiation ability of ES cells to germ cells.3. Wrapped Figlα-ESCs in translucent membrane, transplanted them into renal capsule of reproductive defect mice model, and samppled after 4 weeks. The hematoxylin-eosin (HE) staining and immunofluorescence staining showed that: transplanted Figlα-ESCs could survive in the recipient mice and expressed germ cell specific markers like VASA, SCP3 and Figlαunder the induction in the microenvironment in mice.