Differentiation of Mouse Embryonic Stem Cells Into Gonadotrope-like Cells In Vitro

Abstract

This research was conducted to investigate the potential of mouse embryonic stem (ES) cells to differentiate in vitro into gonadotropes. Undifferentiated ES cells were maintained on mitomycin C-inactivated fibroblasts in the presence of leukemia inhibitory factor (LIF). By a 5-day hanging drop culture devoid of them, ES cells were induced to form multidifferentiated structures called embryoid bodies (EBs). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze gene expression of gonadotrope markers in EBs at different time points during the culture. Homeo box gene expressed in ES cells (Hesx1), LIM homeobox protein 3 (Lhx3), paired like homeodomain factor 1 (Prop1), GATA binding protein 2 (GATA2), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) mRNAs were detected at day 6 EBs and maintained throughout the culture to day 56. FSHbeta and LHbeta proteins were expressed in EBs from day 6 onward. Immunofluorescent labeling of FSHbeta and LHbeta showed that specific staining was restricted to the cytoplasm of some differentiated EB cells. With the prolongation of EB culture, the number of positive cells increased significantly. Both monohormonal and bihormonal cells were present, mainly in clusters within EBs and sparsely distributed among the outermost cells surrounding the EBs. These results indicate that mouse ES cells can give rise to mature gonadotrope-like cells in EBs. It also shows that EBs may serve as a novel model system to study the development and function of gonadotropes.