Isolation, expansion and purification of mouse spermatogonial stem cells in an autologous sertoli cell co-culture system

ABSTRACT

This study presents a simple method for isolation, expansion and purification of neonatal mouse Spermatogonial stem cells. We used enzymatic digestion to isolate a cell suspension of spermatogonia and Sertoli cells from neonatal 2-day-old mice. The cells were cultured in DMEM/F12 that contained 10% serum for two weeks. Sertoli and spermatogonia cell characteristics were confirmed by examining for the presence of vimentin and PLZF proteins, respectively. To assess the rate of spermatogonia stem cell expansion, the area and number of colonies were measured during the two weeks of culture. At the end of the second week, we detected spermatogonia cell-specific expressions of the Stra8, Piwill2, DAZL, and Mvh genes. Current results indicated that isolated Sertoli and spermatogonia cells were immunopositive for specific markers. During the culture period, a significant difference was seen in the number and area of Spermatogonial stem cell colonies [P<0.05] at four time points. In addition, Spermatogonial specific gene expression demonstrated that these cells were undifferentiated after two weeks of culture. Our study showed that co-culture of spermatogonia and Sertoli cells from same source provides a convenient and efficient environment. This co-culture, without the addition of external growth factors and chemical manipulations, can be used for proliferation of spermatogonia stem cells