Objective: Along the biological understanding of spermatogenesis, a major concern is improving the efficiency of the various cultural conditions and evaluation of the safety of the clinical application. Since the interaction between SSCs and their nich is capable of inducing self-renewal in SSCs, suggesting that this signaling pathways are promoted in culture of SSCs, by co-culture with normal sertoli obtained from healthy persons Materials and Methods: Testicular cells isolated from human testis biopsies by two step enzymatic digestion and plating methods. The identity cells were confirmed through immunocytochemistry. We were designed various culture system: co-culture with patient own Sertoli cells, co-culture with normal sertoli cells obtained from a person with normal spermatogenesis and culture of SSC on un-coated dishes. Colonization was evaluated during the 3 weeks of culture. The expression of the α6 and β1 integrins and PLZF as germ stem cell specific markers and nanog, c-myc and oct-4 as pluripotency markers were assessed using quantitative RT-PCR on 2nd and 3rd weeks of culture. Results: Our result showed higher mean in number and diameter of colonies in co-culture groups in the compare with control group. Our data was shown the higher expression of germ stem cell markers during the culture and lower expression of nanog in 3rd weeks in co-culture with patient own Sertoli cells. Co-culture with normal sertoli cells in revealed significant higher expression of PLZF. At 3rd weeks, there are significantly increased in expression profile of α6 and β1 integrins and PLZF in compare to our control group. Co-culture with normal sertoli showed higher expression of germ stem cell specific markers at 3rd weeks of culture versus co-culture of SSCs with the patient own sertoli. Conclusion: Co-culture of human SSCs with sertoli cells has emerged as a suitable method for the enrichment of spermatogonial germ cells.