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You have accessJournal of UrologyInfertility: Therapy1 Apr 2013

1884 CHARACTERIZATION OF IN VITRO PROPAGATED HUMAN SPERMATOGONIAL STEM CELLS (SSCS) PRIOR TO AUTOTRANSPLANTATION

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    INTRODUCTION AND OBJECTIVES

    Over the last three decades effective cancer treatments have improved the survival rates for many types of cancer. Especially in childhood cancer the 5-year event-free survival rates are now around 80%. It is currently estimated that 1 in 250 young adults between 20 and 29 years is a long term survivor of childhood cancer. This implies that adverse side effects including gonadal failure and subsequent poor long-term reproductive outcomes have now become important sequels of previous exposure to chemotherapy and/or radiotherapy. The ensuing sterility can not be prevented or otherwise counteracted, and there are currently no means to preserve fertility prior to treatment in prepubertal boys. This is in contrast to adult men, for whom ejaculated sperm can be cryopreserved and in contrast to women and girls in whom cryopreservation of oocytes or ovarian cortical strips prior to the initiation of cancer treatment can be performed. Recent improvements in Spermatogonial Stem Cell (SSC) technology paved the way to preserve fertility in high risk patients, especially sterile cancer survivors. In vitro propagation of SSCs and characterization of propagated cells are necessary for successful SSCs autotransplantation and consequently restoring fertility.

    METHODS

    In this study we received human testicular tissues from 3 brain death individuals via National Disease Research Interchange (NDRI) 19-27.5 hours after organ recovery. The tissues were immediately cryopreserved when received at our institute. After thawing and enzymatic digestion of the testicular tissue, the testicular cells were seeded in supplemented Stempro medium. The presence of spermatogonial cells and their quantity were evaluated by germ line stem cell (GSC) formation, reverse transcriptase (RT) PCR and flow cytometry.

    RESULTS

    The first GSC cluster formation was seen after 2 weeks of culture. RT PCR for PLZF (ZBTB16), UCHL1 (PGP 9.5), FGFR3 and CD9 as markers for undifferentiated spermatogonial cells (includes SSCs) confirmed the presence of these cells during the culture period (up to 90 days). Flow cytometric analyses showed various percentages (0.7-6%) of HLA-ABC negative/FGFR3 positive cells (Spermatogonial cells) in different passages during the culture period.

    CONCLUSIONS

    Optimization of this SSCs in vitro culture system and adaptation to GLP/GMP protocols are necessary before clinical application of SSC autotransplantation.

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