RRC ID 44728
Author Aoshima K, Baba A, Makino Y, Okada Y.
Title Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement.
Journal PLoS One
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.
Volume 8(10)
Pages e77715
Published 2013-1-1
DOI 10.1371/journal.pone.0077715
PII PONE-D-13-16269
PMID 24204931
PMC PMC3810131
MeSH Adult Stem Cells / cytology* Adult Stem Cells / metabolism Adult Stem Cells / physiology* Animals Cell Culture Techniques / methods* Cell Cycle / genetics Cell Cycle / physiology Cell Differentiation / genetics Cell Differentiation / physiology Cells, Cultured Culture Media / metabolism Culture Media, Serum-Free / metabolism* Fibroblast Growth Factor 2 / genetics Fibroblast Growth Factor 2 / metabolism Fibroblasts / metabolism Gene Expression / genetics Glial Cell Line-Derived Neurotrophic Factor / genetics Glial Cell Line-Derived Neurotrophic Factor / metabolism Glial Cell Line-Derived Neurotrophic Factor Receptors / genetics Glial Cell Line-Derived Neurotrophic Factor Receptors / metabolism Mice Mice, Inbred C57BL Mice, Inbred ICR Spermatogenesis / genetics Spermatogenesis / physiology*
IF 2.74
Times Cited 33
Human and Animal Cells STO(RCB0536)