[Frontiers in Bioscience 11, 2614-2622, September 1, 2006]

In vitro propagation of spermatogonial stem cells from KM mice

Yu-Qiang Shi 1, Qing-Zhong Wang 1, 2, Shang-Ying Liao 1, Yan Zhang 1, Yi-Xun Liu 1, and Chun-Sheng Han 1

1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China, 2 Key Laboratory of Animal Resistance, College of Life Science, Shandong Normal University, Jinan 250014, Shandong Province, China


1. Abstract
2. Introduction
3. Materials and methods
3.1. Experimental animals
3.2. Culture conditions
3.3. Isolation and collection of mouse testis cells
3.4. Differential adherence selection and culture procedures of SSCs
3.5. RT-PCR analysis of marker gene expression
3.6. Immunocytochemical staining and confocal laser microscopic observation of SSCs
3.7. Flow cytometric analysis (FCA) of cells
4. Results
4.1. An improved procedure for SSC isolation and culture
4.2. Differential adherence selection for male germ cells is a simple but effective approach
4.3. Culture and phenotypic characterization of SSCs from KM pup mice
4.4. Presence of Oct4-Sox2 regulatory complex in SSCs
4.5. LIF is not required for in vitro proliferation of SSCs
5. Discussions
6. Acknowledgements
7. References


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.