title: Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement.
abstract: Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology.
authors.affiliation: Laboratory of Pathology and Development, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ; Career-Path Promotion Unit for Young Life Scientists, Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Yoshida Konoe Cho, Sakyo-ku, Kyoto, Japan ; Division of Molecular Pathobiology, Center for Zoonosis Control, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Aoshima
authors.forename: Ai
authors.initials: A
authors.lastname: Baba
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Makino
authors.forename: Yuki
authors.initials: Y
authors.lastname: Okada
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D013091:Spermatogenesis / Q000235:genetics / Q000502:physiology*
chemical_list: D003470:Culture Media; D016895:Culture Media, Serum-Free; C494988:Gfra1 protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D016222:Fibroblast Growth Factor 2