A Preliminary Study On Isolation, Long-term Culturing And Induced Differentiation Of Mouse Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs) have the abilities of self-renewal and differentiation into sperm throughout the life of male animals. They are the only adult stem cells that transmit genetic information to subsequent generations. The establishment of in vitro propagation culture systems and their differentiation research provide technical support for the preservation of the mammalian genetic information and experimental evidence for the mechanism of the spermatogenesis. At present, the separation and long-term culture of mouse spermatogonial stem cells(mSSCs) in vitro is technically mature, but the methods are both complicated and expensive, and different laboratories may use different methods, bringing much inconvenience in the study of SSCs. It is essential to establish a simple and effective method for the isolation and propagation of SSCs further study.Based on the research abroad and repeatedly groped, we have established a successful mSSC primary isolation and culture method for Sertoli cells and mSSCs cocultivation. This method is easy to operate and have a high clone formation rate, and the mSSC grows fast. There are many grape bunches of clone formation in two days. More importantly, this method in the process of mSSCs isolation and culture don’t need to use StemPro- 34 stem cell culture medium, many additives and various growth factors. It is only need the conventional medium DMEM sugar and three kinds of growth factors such as GDNF, bFGF. The method has established the long-term culture system from kunming mice and C57 BL / 6 mice from background of non-DAB / 2.This method that established mSSC cells line could be stable grown on mouse embryonic fibroblast(mouse embryonic fibroblast, MEF) or Sertoli cells as feeders. The culturing cells were identified as mSSCs by alkaline phosphatase staining, RT-PCR and indirect immunocytochemistry. The mSSCs were induced to differentiate into haploid germ cells with retinoic acid(RA). RT-PCR, indirect immunocytochemistry and flow cytometry(FCM)were used to identify the haploid germ cells. The results showed that the mSSCs could be induced to differentiate into haploid germ cells with RA in vitro.This long-term culture system is suitable for mSSCs from EGFP transgenic mice and Kunming mice, simplifies the separation steps and reduces the culture cost, laying the foundations for subsequent studies on the mechanism of RA-induced.The method established for separation and culture of mSSC is easy to operate and reduce the cost. It provides a good technical support for subsequent spermatogenesis mechanism research and SSCs separation and culture of other species.