AB193. Isolation and long-term culture of human spermatogonia stem cell

OBJECTIVE: In vitro culture of spermatogonia stem cell would be benefit to the research on spermatogenesis mechanism. For those who suffer from azoospermia with a spermatogenesis arrest, there would be a hope to gain functional gamete via inducing their own spermatogonia stem cells if we could achieve long-term culture of these cells in vitro. However, in spite of that the rodent spermatogonia stem cells had been cultured in vitro over months successfully, there was few report on the long-term culture of human spermatogonia stem cells, which was mainly due to the fragile of human spermatogonia cells and the critical culture condition essential for them. This work aimed to explore a gentle way to isolate human spermatogonia stem cells and achieve a long-term in vitro culture of them. DESIGN: Exploratory research. MATERIALS AND METHODS: Human testis tissues of obstructive azoospermia patients were obtained via surgery and digested into cell suspension via two-step enzymatic digestion. Matrix selection was applied to isolate the human spermatogonia stem cells. SG medium was used as the condition medium for human spermatogonia stem cell culture. Immunofluorescence was carried out to characterize the isolated cells. RESULTS: The isolated cells survived over 3 weeks in vitro with a good morphology that was identical with human spermatogonia stem cells and some of them former colonies. Immunofluorescence showed that over 90% isolated cells expressed the spermatogonia stem cell markers such as GPR125, UCHL1, PLZF and GFRα-1. CONCLUSIONS: Matrix selection would be a good way to isolated human spermatogonia stem cells and SG medium was suitable for the long-term culture of these cells. Improvements should be made for a longer period of culture and a higher purity. SUPPORT: This work was supported by China National Key Project (2010CB945200 to ZL), a key grant from National Nature Science Foundation of China (31230048 to ZH), and grants from National Science Foundation of China (31171422 to ZH)