GFRA1 and RET are co-receptors for GDNF and they have been regarded as markers for SSCs30,31,32. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine. Spermatogonial stem cells (SSCs) are a subpopulation of type A spermatogonia. Studies on SSCs are of unusual significance in view of their unique characteristics1. Firstly, SSCs are the only adult stem cells that transmit genetic information to subsequent generations, and thus they represent an invaluable resource for experimental modification of the mammalian genome2. Secondly, SSCs self-renew throughout mammalian whole life and they differentiate into Salubrinal spermatocytes and mature spermatozoa, and therefore they can be utilized as an excellent model to uncover the molecular mechanisms underlying the renewal versus differentiation of stem cells. Salubrinal Thirdly, it might be feasible to use SSC transplantation to restore fertility in cancer patients after chemotherapy and/or irradiation therapy3. Male infertility has become a major health and interpersonal concern worldwide, due to environmental factors, inflammation, and anti-tumor therapy4. It has been reported that infertility affects around 15% of couples and male factors account for 50%5. Azoospermia has been found in 1% of the general populations and it comprises 10C15% male infertility6. We have recently shown that human SSCs can be induced to differentiate into haploid spermatids with fertilization and developmental capacity7, reflecting that they can provide mature and functional male gametes for azoospermic patients with SSCs. Lastly and more importantly, a Salubrinal number of studies have exhibited that SSCs can acquire pluripotency to become embryonic stem (ES)-like cells that are able to differentiate into all cell lineages of three germ cell layers8,9,10,11,12,13. Strikingly, numerous studies by peer and us have recently exhibited that SSCs are able to directly transdifferentiate into the cells of other lineages both and and a long-term culture system of human SSCs has not yet been established. The limited life-span and rare number of human SSCs represent a serious problem for understanding molecular mechanisms of human spermatogenesis; and iv) there is not yet a human SSC line to obtain sufficient cells for their usage from the bench to bed side. Spermatogonial cell line and SSC line have been set up using plasmids over-expressing telomerase or SV40 large T antigen in rodents19,20. Nevertheless, a human SSC line is currently unavailable. Notably, there are distinct identity and cell types for rodent and human SSCs, since the As spermatogonia are Salubrinal the actual stem cells for rodents, while the Adark and Apale spermatogonia are generally regarded as human SSCs. Moreover, the phenotypic characteristics between rodent and human SSCs are different. As examples, OCT-4 (also known as POU5F1) is usually a hallmark for mouse SSCs, whereas it is absent in human SSCs18. Therefore, the mechanisms regulating fate decisions of human and rodent SSCs are distinct. Here we have for the first time reported Rabbit Polyclonal to Smad1 a human SSC line by stably introducing SV40 large T antigen through lentivirus contamination. Cellular, molecular, and functional assays and revealed that this cell line was human SSCs without Y chromosome microdeletions of numerous genes or tumor formation and it could be expanded with significant increases of cell number for over one and half years and colonized in the recipient mice. Significantly, our ability of establishing human SSC line could offer an unlimited.