Bone morphogenetic protein 4 role on in vitro differentiation of primordial germ cells from mouse embryonic stem cells

摘要

Background: The purpose of this study was to determined optimal dose of BMP4 and its time period on differentiation Promordial Germ Cells (PGCs) from mouse Embryonic Stem Cells (mESCs) in vitro. Methods: To differentiation of PGCs, EBs from mouse ES cells were cultured in concentrations 0, 5, 10 and 50 ng/ml BMP4 at the different time interval. Viability of PGCs was assayed by MTT. Also, germ cell markers Oct-4, Stella and Mvh were analyzed by flow cytometry, Immunocytochemistry, electrophoresis gel and Real Time PCR. Results: BMP4 at 10 ng/ml concentrations for day 4 had the best on the viability. The data of flowcytometry demonstrated most of Mvh+ cells was observed in D4B10. Immunocytochemistry of EBs in D2B5, D2B10, D4B5 and D4B10 groups identified cells that are positive to Mvh. The result of Real time PCR was illustrated that expression of gene Oct-4 in the control groups was high and it gradually decreased with adding concentrations 5 ng/ml and 10 ng/ml of BMP4 on days 2nd, 4th and 6th. The maximum expression of Stella was observed in D4B10 group. However, the expression of Stella with BMP4 concentration of 50 ng/ml on the day 2nd, 4th and 6th considerably were decreased. The expression of Mvh with BMP4 concentrations of 0 ng/ml and 50 ng/ml was none. However, the expression of Mvh were increased in D2B5, D2B10, D4B5 and D4B10 groups (not significantly). Conclusions: The results propose that concentrations of 10 ng/ml BMP4 at days 4th had the optimal effects on differentiation of PGCs to mESCs.