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Journal: یاخته
Year:2010 | Volume: | Issue:
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Title

ORAL PRESENTATIONS; PROPAGATION OF HUMAN SPERMATOGONIAL STEM CELLS IN VITRO

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 Start Page 16 | End Page 16

Abstract

 Objective: Recently developed, highly effective, cancer therapy for children allows the majority of them to survive their cancer. One of the major side effects of cancer therapy in male patients is sterility. There are currently no means to preserve reproductive potential in prepubertal boys, which contrasts with adolescents and adults, for whom cryopreservation of semen prior to chemotherapy or radiotherapy is available and widely used. Therefore, establishing a human spermatogonial STEM cell (hSSC) culture system to allow successful autotransplantation for young boys diagnosed with cancer is of utmost importance. As the final number of transplantable cells will influence the success rate of this technique, we first focus on the propagation of hSSC in culture.Materials and Methods: We used testicular tissue from six men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were isolated with a two steps enzymatic digestion and overnight differential plating. Testicular cells were cultured in supplemented STEMPro medium. Some formed germline STEM cell (GSC) clusters were taken out of the culture and subcultured on human placental laminin coated dishes. The presence of spermatogonia in the cultures was determined by immunohistochemistry and RT-PCR for spermatogonial markers (integrin-α6, integrin-β1, PLZF). The spermatogonial STEM cell transplantation assay was performed using busulphan treated nude mice, as the functional test of STEM cell capability. Human cells in recipient mouse testis were detected by Fluorescent In situ hybridization (FISH) using the most common human specific repetitive DNA sequence (COT) as a probe.Results: Germline STEM cell (GSC) cluster formation was observed in the testicular cell cultures of all six men in testicular cell cultures and in subcultures GSCs. Testicular cells and subcultured GSCs could be cultured for at least 15 and 28 weeks respectively, while expression of spermatogonial cell surface markers integrin-α6 and integrin-b1 (on RNA level) and spermatogonial nuclear marker PLZF (on RNA and protein levels) was maintained. The mouse transplantation assay showed successful colonization of cultured testicular cells in 4 out of 6 patients and from the subcultured GSCs in 1 out of 2 patients, indicating the presence of functional spermatogonial STEM cells. By determining the number of colonies of transplanted cultured cells from early and later passages of the same culture, we found a more than 50 fold increase of hSSC in 19 days in our testicularcell culture when cultured from day 28 to 47 (passage 2 to 5) and a more than 18000 fold increase in number of hSSC in 64 days in our subcultured GSCs when cultured from day 77 to 141 (passage 7 to 12).Conclusion: This report outlines the first successful long term culture and proliferation of hSSC in vitro. This is an important step forward to future clinical application of SSC autotransplantation in prepubertal boys diagnosed with cancer to preserve their fertility .

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