Studying On Effective Isolation And Propagation Of Spermatogonial Stem Cells In Vitro

Spermatogonial stem cell(SSCs) provide the foundation for spermatogenesis throughoutthe whole male life. It can renew itself and produce the destined differentiatedspermatogonium, and is the only one stem cell who can transmit genetic information tooffspring in male adult creation. We ordinarily carried out the cultures using a feeder layer ofmouse embryonic fibroblasts (MEFs), STO cells, or co-culture with Sertoli cells and theisolated SSCs, with added some growth factor such as GDNF, bFGF, EGF and LIF. In ourstudy, we try to establish a convenient and effective method for isolating and propagating themale germline stem cells in vitro. So we isolated the germline stem cells of the testes froman adult mouse, a6-day-old mouse and a2-month-old white Suffolk by two-step digestion,After selection morphologically, we cultured the cells on the purified Sertoli cells withoutLeydig cells, MEFs or SEFs as feeder respectively. We tested the efficiency of the differentculture systems, and established the male germline stem cell lines from both mouse and sheep.We found that the efficiency of propagation of SSCs from adult mouse on Sertoli feederwithout GDNF and bFGF is comparable to the one on MEFs with GDNF and bFGF. Theefficiency of establishment is quite high enough via this method. Especially the SSCs from6-day-old mouse can form the three-dimensional structure in this system, and the SSCs from2-month-old white Suffolk grows well in this system too. Our study guaranteed to achieve thesingle genotype, and reduced the cost of culturing adult male germline stem cells in vitro.This method provided the foundation for making the transgenic animals and disease modelsderived from adult genotype.