[Establishment of a long-term culture system for mouse spermatogonial stem cells in vitro].
OBJECTIVE
To establish a long-term proliferation culture system for mouse spermatogonial stem cells.


METHODS
Testis tissues were obtained from 30 newborn male ICR mice on postnatal day 2-6. Testis cell suspension was collected by two-step enzymatic digestion prior to culture. The dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium on mouse embryonic fibroblasts (MEF) feeders. Their proliferation was determined by the BrdU incorporation test and the cultured cells identified by alkaline phosphatase (AP) activity, immunofluorescence staining and RT-PCR assay.


RESULTS
The cultures remained in a steady state and continued to generate germ cell colonies. The undifferentiated state was confirmed by strong positivity for AP activity, immunofluorescent staining of GFRalpha-1+ /Oct-4+ /VASA+ /SCP3- and GFRalpha-1+ /Oct-4+/SCP3- at the gene expression levels.


CONCLUSIONS
Mouse spermatogonial stem cells could be expanded in our defined culture system and passaged steadily in vitro. The harvested cells remained in an undifferentiated state, which has provided a good platform for the study of spermatogenesis in vitro.
Sun
Yuan
Y
Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha, Hunan 410078, China. sdsunyuan@yahoo.com.cn
Tao
Ke
K
Tu
Jiong-Jiong
JJ
Zhu
Wen-Bing
WB
Fan
Li-Qing
LQ
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors
D000469:Alkaline Phosphatase