[Long-term culture and identification of spermatogonial stem cells from BALB/c mice in vitro].
OBJECTIVE
To establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation.


METHODS
Testis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis.


RESULTS
After 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia.


CONCLUSIONS
SSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.
Shen
Fu-Jin
FJ
Department of Obstetrics and Gynecology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China.
Zhang
Ci
C
Yang
Si-Xing
SX
Xiong
Yun-He
YH
Liao
Wen-Biao
WB
Du
Xian-Jin
XJ
Wang
Ling-Long
LL
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*