In vitro propagation of spermatogonial stem cells from KM mice.
Spermatogonial stem cells (SSCs) are a unique type of stem cells in that they transmit genetic information to the next generation by producing sperms. Studies of SSC proliferation and differentiation have been hampered by the inability of reconstructing these processes in vitro, particularly in a serum-free culture system. Several groups have reported the long term culture of SSCs during which SSCs self-renew and restore spermatogenesis when transplanted back to recipient testes. However, different protocols and mice with particular genetic background have been used by different laboratories, and the techniques have not been adopted widely. In the present study, we first established a SSC isolation and culture system composed of differential adherence selection of SSCs, serum-free medium and mouse embryonic fibroblast (MEF) feeder cells. SSCs from KM pups could be cultured on MEF feeders in StemPro-34 SFM Medium supplemented with glial cell line-derived neurotrophic factor (GDNF), soluble GDNF family receptor alpha-1 (GFRa1) and basic fibroblast growth factor (bFGF) for 1 month. These SSCs were characterized morphologically and by examining the expression of marker genes. Expression of Oct4 and Sox2, which are crucial factors in embryonic stem cell (ESC) self-renewal, were detected in our cultured SSCs, suggesting that SSCs may share with ESCs some common mechanisms in self-renewal regulation. We also found that LIF had no effect on the proliferation of cultured SSCs derived from KM mice.
Shi
Yu-Qiang
YQ
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Wang
Qing-Zhong
QZ
Liao
Shang-Ying
SY
Zhang
Yan
Y
Liu
Yi-Xun
YX
Han
Chun-Sheng
CS
D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors; D013093:Spermatogonia / Q000254:growth & development; D015534:Trans-Activators / Q000096:biosynthesis
D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D015534:Trans-Activators