Derivation of ES-like cell from neonatal mouse testis cells in autologous sertoli cells co-culture system

Spermatogonial stem cell [SSC] is a self-renewing population of male adult stem cell. SSCs have a differentiation potential which are similar to embryonic stem cells. These Embryonic stem like [ES-like] cells can be a potential source for pluripotent cells for stem cell-based therapy. This study presents an economical and simple co-culture system for pluripotent stem cells generation from neonatal mouse testis. Isolated testicular cells were cultured in DMEM/F12. Characteristics of the isolated cells and obtained ES-like cell were immune-cytochemically confirmed by examining the presence of PLZF, vimentin, Oct4 and Nanog protein. Expression of the pluripotency and germ-cell specific genes was analyzed by qPCR in derived ES-like colony and SSCs respectively. The experiment results indicated that our method of obtaining pluripotent ES-like cells from spermatogonial cells [SCs] is simpler than the described methods. ES-like cells were immunopositive for pluripotency markers. ES-like cell qPCR results indicated significant increase in pluripotency genes expression and significant decrease in germ cell-specific genes expression. The results indicated that ES-like cell with pluripotency characteristic were generated from freshly isolated spermatogonial cells. The pluripotent stem cells provide a cellular reservoir usable for regenerative medicine instead of embryonic stem cells.

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

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