Effect of Different Concentrations of Forskolin Along with Mature Granulosa Cell Co-Culturing on Mouse Embryonic Stem Cell Differentiation into Germ-Like Cells

Germ cell development processes are influenced by soluble factors and intercellular signaling events between them and the neighboring somatic cells. More insight into the molecular biology of the germ cell development from embryonic stem (ES) cells and investigation of appropriate factors, specifically those targeting differentiation processes, is of great importance. In this study, we established an in vitro model with higher ES cell differentiation rate to germ cells, using adenylate cyclase activator, forskolin.

ES cells were first cultured for five days, leading to embryoid body (EB) formation. Subsequently, the EB were dissociated and cultured for an additional three days in different forskolin concentrations of 5, 20, and 50 µM, with or without granulosa cells (GC) co-culture. On the 8th day, we analyzed the expressions of 5 germ cell-specific markers using quantitative real-time-PCR technique along with cell viability assay by MTT test.

Our results showed that in the GC-free cultures, a 50-µM concentration of forskolin resulted in a significant increase in Mvh, Gdf9, Scp3, and Rec8 expression levels in comparison to the control. However, when the cells were co-cultured with the GCs, 20-µM concentration of forskolin could also increase the expression of those germ cell-specific marker genes. Furthermore, results from the MTT assay showed enhanced cell proliferation and survival at all three concentrations of forskolin, but 20-µM concentration was the most potent one.

These data indicate that forskolin can stimulate differentiation and proliferation, dose-dependently; however, the influence of GCs co-culturing should not go unnoticed.