Context Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility. Objective To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation. Design, Setting, and Participants Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation. Main Outcome Measures Propagation of spermatogonial stem cells over time. Results Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18 450-fold within 64 days in the germline stem cell subculture. Conclusion Long-term culture and propagation of human spermatogonial stem cells in vitro is achievable.

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

Sertoli cells isolated from the adult mouse and human testis resume proliferation in culture. After 20 days of culture in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12) medium containing 5%% fetal calf serum, about 36%% of the mouse Sertoli cells, identified by their immunohistochemical staining for the Sertoli cell marker vimentin, incorporated bromodeoxyuridine (BrdU). The renewed proliferation was associated with a 70%% decrease in expression of the cell cycle inhibitor CDKN1B (P27(kip1)) and a 2-fold increase in the levels of the proliferation inducer ID2. In vivo, the balance between cell cycle inhibitors and inducers probably is such that the cells remain quiescent, whereas in culture the balance is disturbed such that Sertoli cells start to proliferate again. The renewed proliferative activity of Sertoli cells in culture was further confirmed by double staining for BrdU and the Sertoli cell marker clusterin (CLU), showing about 25%% of the CLU-positive Sertoli cells to be also positive for BrdU after 13 days of culture. Radiobiologically, Sertoli cells are also different from other quiescent somatic cells in the testis because they express several DNA repair proteins (XRCC1, PARP1, and others). Indeed, a comet assay on irradiated Sertoli cells revealed a 70%% reduction in tail length and tail moment at 20 h after irradiation. Hence, Sertoli cells repair DNA damage, whereas other quiescent somatic testicular cells do not. This repair may be accomplished by nonhomologous end joining via XRCC1 and PARP1. In conclusion, cell kinetic and radiobiological data indicate that Sertoli cells more resemble arrested proliferating cells than the classic postmitotic and terminally differentiated somatic cells that they have always been assumed to be.

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In recent years, embryonic stem (ES) cell-like cells have been obtained from cultured mouse spermatogonial stem cells (SSCs). These advances have shown that SSCs can transition from being the stem cell-producing cells of spermatogenesis to being multipotent cells that can differentiate into derivatives of all three germ layers. As such, they offer new possibilities for studying the mechanisms that regulate stem cell differentiation. The extension of these findings to human SSCs offers a route to obtaining personalized ES-like or differentiated cells for use in regenerative medicine. Here, we compare the different approaches used to derive ES-like cells from SSCs and discuss their importance to clinical and developmental research.

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