O-19: Proliferation of Small Population of Spermatogonial Stem Cells in Azoospermic Patients

Background: With treatment success in young boys with cancer, long-term effects of cancer treatment have found importance in pediatric oncology. Temporary or permanent infertility after treatment is an important subject in childhood and adult cancer patients which decrease quality of life. The one approach to overcome infertility in these cases is to cryopreserve small biopsy testicular tissue before chemotherapy and to propagate and autotransplant spermatogonial stem cells from this tissue after cancer survival. So, culturing and the access to sufficient numbers of spermatogonial stem cells (SSC) in vitro is nessecery for increasing chances of efficient transplantation. The aim of this study was to compare the in vitro effects of laminin and growth factors on the proliferation of adult human spermatogonial stem cells. Materials and Methods: After using TESTE samples in Embryology lab, the remainders of testes tissues were used for cell isolation. Testicular cells were isolated by two steps of enzymatic digestion and cultured in DMEM supplemented with 5% FCS. During the culture, isolated spermatogonial cells were treated either with various concentrations of GDNF 40 ng/ml, FGF 10 ng/ml, EGF 20 ng/ml, LIF 10-3 M or with human placental laminin-coated dishes with the same growth factors. Colony assay was performed by means of a light microscopy during culture. Presence of spermatogonia was determined by Ultrastructure study of cell colonies, reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers ( PLZF, Mvh- VASA, DAZL, Igα6 and Igβ1). The presence of functional spermatogonial stem cells in culture was confirmed through xenotransplantation to testes of azoospermic mice. The migrated human spermatogonial stem cells after transplantation were detected by Anti-BrdU fluorescence. The statistical significance between mean values was determined using statistical tests. Results: The results indicated that treatment with the growth factors is essential for in vitro colonization of adult human spermatogonial cells. cultivation with growth factors on human placental laminin-coated dishes or without human placental laminin-coated dishes showed a significant increase in the number and diameter of colonies compared in treated and control groups. Also, Expression of spermatogonial markers on both the RNA and protein levels were maintained throughout the entire culture period. A transplantation experiment, using azoospermic mice with Busulfan showed the presence of SSC among the cultured cells. In addition TEM study strongly suggested typical morphology spermatogonial cells present among the culture cells. Conclusion: We conclude that human SSCs are self-renewed in our culture system and this system can be used and be viable for the propagation of these cells from small biopsy.