Long-term culture and identification of spermatogonial stem cells from BALB/c mice in vitro / 中华男科学杂志
National Journal of Andrology
; (12): 977-981, 2008.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-309775
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Espermatogônias
/
Células-Tronco
/
Técnicas de Cultura de Células
/
Biologia Celular
/
Métodos
/
Camundongos Endogâmicos BALB C
Tipo de estudo:
Estudo prognóstico
Limite:
Animais
Idioma:
Chinês
Revista:
National Journal of Andrology
Ano de publicação:
2008
Tipo de documento:
Artigo