Improved serum- and feeder-free culture of mouse spermatogonial stem cells Running title: Serum-free culture of spermatogonia

Spermatogonial stem cells (SSCs) undergo self-renewal division, which can be recapitulated in vitro. Attempts to establish serum-free culture conditions for SSCs have met with limited success. Although we previously reported that SSCs can be cultured without serum on laminin-coated plates, the growth rate and SSC concentration were relatively low, which made it inefficient for culturing large numbers of SSCs. In this study, we report on a novel culture medium that showed improved SSC maintenance. We used Iscove modified Dulbecco medium, supplemented with lipid mixture, fetuin and knockout serum replacement. In the presence of glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), SSCs cultured on laminin-coated plates could proliferate for more than 5 months and maintained normal karyotype and androgenetic DNA methylation patterns in imprinted genes. Germ cell transplantation showed that SSCs in the serum-free medium proliferated more actively than those in the serum-supplemented medium and that the frequency of SSCs was comparable BOR Papers in Press. Published on September 10, 2014 as DOI:10.1095/biolreprod.114.122317 Copyright 2014 by The Society for the Study of Reproduction.