Comparison of different in vitro differentiation conditions for murine female germline stem cells.

OBJECTIVES: In vitro differentiation of oocytes from female germline stem cells (FGSCs) has exciting potential applications for reproductive medicine. Some researchers have attempted to reveal the in vitro differentiation capacity of FGSCs. However, no systematic comparative study of in vitro differentiation conditions has been performed for murine FGSCs (mFGSCs). MATERIALS AND METHODS: mFGSCs line was cultured under five different conditions for in vitro differentiation. RT-PCR was performed to detect the expression of Oct4, Fragilis, Blimp1, Mvh, Scp3 and Zp3. Immunofluorescence was carried out to test the expression of Mvh, Fragilis and Zp3. Two-photon laser-scanning microscope was used to analyze nucleus-plasma ratio, and the proportion of chromatin of GV oocytes differentiated from mFGSCs in vitro (IVD-GVO), GV oocytes from in vivo (GVO) and mFGSCs. RESULTS: RT-PCR and immunofluorescence showed that mFGSC line expressed germ cell-specific markers, but not a meiosis-specific marker. By evaluating five different in vitro differentiation conditions, condition 5, which included a hanging drop procedure and co-culture of mFGSCs with granulosa cells, was shown to be optimal. mFGSCs could be successfully differentiated into germinal vesicle (GV) -stage oocytes under this condition. 3D observation revealed that both the nucleus-plasma ratio and proportion of chromatin were not significantly different between IVD-GVO and GVO. CONCLUSION: We evaluated five in vitro differentiation conditions for mFGSCs and successfully differentiate mFGSCs into GV-stage oocytes using a three-step differentiation process.

Conversion of female germline stem cells from neonatal and prepubertal mice into pluripotent stem cells.

Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource for cell-based therapies. However, therapies using these cells will only benefit males but not females. Recently, female germline stem cells (FGSCs) were discovered in ovaries. Whether FGSCs can be converted into pluripotent stem cells, similar to spermatogonial stem cells, is unknown. Here, we demonstrate that female embryonic stem-like cells (fESLCs) can be generated within 1 month from the stably proliferating FGSCs cultured in embryonic stem cell (ESC) medium. fESLCs exhibit properties similar to those of ESCs in terms of marker expression and differentiation potential. Thus, our findings suggest that generation of patient-specific fESLCs is feasible and provides a foundation for personalized regenerative applications.