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Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis. Although several in vitro SSC culture systems have been developed, these systems include serum or fibroblast feeders, which complicate SSC self-renewal analyses. Here we developed a serum and feeder-free culture system, in which SSCs propagated for long-term. In addition to the SSC self-renewal factors including glial cell line-derived neurotrophic factor (GDNF), supplementation with fetuin and lipid-associated molecules was required to drive SSC proliferation in vitro. Cultured cells proliferated for at least 6 months at a rate comparable to that of serum-supplemented cultured cells. However, germline potential was reduced under serumand feeder-free conditions, because we observed a lower SSC frequency after germ cell transplantation. Nevertheless, the cultured cells completed spermatogenesis and produced offspring following spermatogonial transplantation into seminiferous tubules of infertile mice.+ p( g; i% Y( q8 T$ d4 i8 m
This culture system provides a basic platform for understanding the regulation of SSC fate commitment in vitro and for further improving SSC culture medium.
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Address correspondence and reprint requests to: Takashi Shinohara, Department of Molecular5 ]0 U+ }9 K0 ~; }$ L8 l) A3 O; V
Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto) H0 X6 `' A i- g5 w; Q4 |/ |" e
606-8501, Japan. R7 i# V% Z- N9 N9 e
Tel: 81-75-751-4160; Fax: 81-75-751-4169; E-mail: tshinoha@virus.kyoto-u.ac.jp5 k6 m7 J% k8 _
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