Construction And Proteomic Analysis Of Spermatogonial Stem Cells (SSCs)

In recent years, infertility has been a serious social problem. The infertility caused by male factor accounts for half of the total munber of patients. The 2010 Nobel Prize in Physiology or Medicine was awarded to the British scientist Robert Edwards, in recognition of his outstanding contribution to the assisted reproductive technologies, especially in the technology of In Vitro fertilization(IVF) therapy. Nowadays, assisted reproductive technology has helped more than 10% couples in the world who are suffering infertility to get their own children. However, due to the technical limitations, assisted reproductive techniques, including IVF, intracytoplasmic sperm injection(ICSI) and so on, require both spouses to provide healthy gametes—eggs and sperms. The azoospermia patients cannot accept this kind of treatment due to absence of mature male gametes. Thus, they have still not be able to get real sense of offspring. Among the all the azoospermia patients, Sertoli Only takes less than 17%. Most of the patients contain normal spermatogonial stem cells(SSCs) in their testis. SSC is a kind of important stem cells in the testis. SSCs can either to self-renew or to form daughter cells that will begin the complex differentiation process of spermatogenesis, resulting in mature spermatozoa. The production of functional male gametes is dependent on the continuous activity of SSCs. Gametes are carriers of genetic information, and are essential for species continuity. Gene editing on SSCs is easy to be passed on the next generation. So SSCs are convient to produce transgenic animal model. Therefore, SSCs have great prospects in the clinical treatment of azoospermia, but there are still many technical difficulties.First, we established three SSCs lines in vitro. The cell lines could undergo long-term proliferation and maintain the function of spermatogenesis when transplanted back to the testis of recipient mice.Then we did some application on them. First, there have not had a method to make SSCs to complete meiosis procedure except transplanting them back to the seminiferous tubules of testis. To humans, transplanting cells cultured for long time in vitro has a lot of safety risks. And this method could not help those testis environment deletion patients. It is urgent to develop new technology which could support SSCs to undergo meiosis independent of existing testicular environment. The biggest difficulty of this technology is that the SSCs niche in vivo could hardly be simulated artificially. In this study, had successfully transplanted the pellet mixed with the long-term cultured SSCs and the somatic cells from neonatal testis under the renal capsule, which could form a testis-like tissue and support the proper spermatogenesis of SSCs. Functional sperm were produced could generate healthy offspring after ICSI. Thus, we rebuilt the construction of seminiferous epithelium ectopically and made the SSCs to spermatogenesis out of existing testicular microenvironment. Next, SSCs were difficult to accept exogenous gene integration and easy to differentiate in culture, so it was hard to do gene editing on SSCs previously. Now, a new technology base on CRISPR-Cas9 system could highly improve the gene targeting efficient. So in this part, we the applied CRIPSR-Cas9 system to successfully conduct gene knockout in SSCs.Finally, though several signal pathways which regulate the self-renewal and differentiation of SSCs were revealed in previous studies. It is also able to maintain their long-term proliferation in culture. The whole regulatory network of SSCs fate decision remains poorly understood. It greatly restricts the clinical application of SSCs. After that, in order to insight the protein expression state in SSCs, we conducted proteomics analysis to the SSCs. As the result, we identified specific surface marker of SSCs which could be used to enrich SSCs from testis. Meanwhile, we speculated biological events the proteins expressed by SSCs take part in through bioinformatics analysis. It provided a basis of better understanding the regulatory networks in the self-renewal and differentiation of SSCs.In conclusion, our study solved the three main technological defects in SSCs applications. It could highly promote the clinical treatment based on SSCs.