A screen of suitable inducers for germline differentiation of chicken embryonic stem cells

Differentiation of germ cells from embryonic stem cells in vitro could have great application for treating infertility and provide an excellent model for uncovering molecular mechanisms of germline generation. In this study, we aim to screen the suitable inducers that may prove the efficiency of driving chicken embryonic stem cells (ES cells) toward germ cells. The male ES cells were separeted into different groups: single retinoic acid (RA) treatment, co-cultured with sertoli cell feeder with RA induction, cultured on matrix proteins (fibronectin, laminin and collagen) with RA treatment, cultured on fibronectin with sertoli cell feeder and RA induction, and single bone morphogenetic protein 4 (BMP4) treatment. Quantitative RT-PCR and immunoourescence were performed to characterize the ES cells differentiation process. The results showed that spermatogonial stem cells (SSCs)-like were not detected in single RA and RA with collagen groups, but were observed in the other groups. The expression of ES specific genes (Nanog and Sox2) was decreased while SSCs marker genes (Dazl, Stra8, integrin α6, integrinβ1 and C-kit) was remarkably increased. The multiple comparsion results showed that the expression of SSCs marker genes in RA with sertoli cells group was significantly higher than the other groups(P<0.05). Collectively, our results suggested that chicken ES cells possess the potency to differentiate into SSCs-like cells in vitro through RA, matrix proteins, sertoli cells and BMP4 induction, of which co-cultured with sertoli cell feeder with RA induction was proved to be the best.