Abstract
BACKGROUND: Stage-specific embryonic antigen-1 (SSEA1) is a cell surface carbohydrate that its pattern expression is changed during induction of mouse embryonic stem cell differentiation. In this study, the spatial distribution of SSEA1 on primordial germ cells differentiation and subsequent progression into oocyte-like cells from mouse embryonic stem cells in vitro was evaluated.
METHODS: Embryoid bodies from mouse embryonic stem cells were cultured for two days with 5 ng/mL BMP4. SSEA1 positive and negative cells were separated using the MACS system and cultured separately in a conditioned medium consist of in vitro maturation medium diluted in DMEM [1:1] for 10 days. We assayed viability, colony formation and alkaline phosphatase activity (ALP) of sorted cells. Also, germ cell markers were analyzed by flow cytometry, Immunocytochemistry and RT-PCR.
RESULTS: Viability percent SSEA1 positive cells were more than SSEA1 negative cells. SSEA1 positive cells and SSEA1 negative cells formed compact and flat colonies respectively. Unlike the SSEA1 positive population, the SSEA1 negative colonies showed a weak ALP activity. SSEA1 positive cells expressed Oct4, Stella, Mvh, c-kit, Scp3, Desmin, GFAP and Albumin. Interestingly, SSEA1 negative cells expressed Desmin and GFAP. The population of Mvh-positive cells in SSEA1 positive was 17.74%. All specific oocyte mentioned genes were detected in the SSEA1 positive. Also, oocyte specific proteins GDF9 and ZP3 were detected using Immunocytochemistry.
CONCLUSION: Our results suggest that conditioned medium provides a suitable niche to differentiation and progression putative primordial germ cells derived from the SSEA1 positive toward oocyte-like cells.