title: Improved serum- and feeder-free culture of mouse germline stem cells.
abstract: 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 mo 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 between the two culture media. Cultured cells underwent germline transmission. Development of a new serum- and feeder-free culture method for SSCs will facilitate studies into the effects of microenvironments on self-renewal and will stimulate further improvements to derive SSC cultures from different animal species.
authors.affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan Japan Science and Technology Agency, PRESTO, Kyoto, Japan.
authors.forename: Mito
authors.initials: M
authors.lastname: Kanatsu-Shinohara
authors.affiliation: RIKEN Bioresource Center, Tsukuba, Japan.
authors.forename: Narumi
authors.initials: N
authors.lastname: Ogonuki
authors.affiliation: RIKEN Bioresource Center, Tsukuba, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Matoba
authors.affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Morimoto
authors.affiliation: RIKEN Bioresource Center, Tsukuba, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Ogura
authors.affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan Japan Science and Technology Agency, CREST, Kyoto, Japan tshinoha@virus.kyoto-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shinohara
mesh_terms: D053687:Adult Stem Cells / Q000502:physiology*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D017690:Cell Transplantation; D003470:Culture Media / Q000737:chemistry*; D016895:Culture Media, Serum-Free; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice
chemical_list: D003470:Culture Media; D016895:Culture Media, Serum-Free; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse
keywords: spermatogenesis; spermatogonia; spermatogonial stem cells; stem cells; testis