[The feeder layer of human embryonic fibroblasts supports the growth of human spermatogonial stem cells].
OBJECTIVE
To investigate the methods and conditions for the isolation, purification and culture of human spermatogonial stem cells (SSCs) on the feeder layer cells of human embryonic fibroblasts (hEFs).


METHODS
SSCs isolated and purified from normal human fetal testicular tissues by sequential two-step enzyme digestion and Percoll uncontinuous density gradient centrifugation were cultured on the feeder layer cells of hEFs isolated from 5-9 weeks old human embryos. The surface markers SSEA-1 and OCT4 of the SSCs were detected by immunohistochemistry; the alkaline phosphatase (AKP) activity of the SSC clones measured; and the expressions of the SSC-related genes determined by RT-PCR.


RESULTS
SSCs survived, proliferated and formed colonies on the feeder layers, and the colonies were highly positive for SSEA-1 and OCT4, with strong AKP activity and high expressions of the SSC-related genes.


CONCLUSIONS
The feeder layer of hEFs supports the growth of human spermatogonial stem cells.
Wang
Yu-Bin
YB
Department of Urology, Shanghai Institute of Andrology, Shanghai, China. wangyb1980@163.com
Chen
Bin
B
Wang
Ying-Chao
YC
Zhang
Zhi-Ling
ZL
Wang
Hong-Xiang
HX
Lu
Yong-Ning
YN
Xiang
Zu-Qiong
ZQ
Hu
Kai
K
Yang
Yi-Ke
YK
Han
Yin-Fa
YF
Wang
Zheng
Z
Wang
Yi-Xin
YX
Huang
Yi-Ran
YR
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology*; D006801:Humans; D008297:Male; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*