Improved Serum- and Feeder-Free Culture of Mouse Germline Stem Cells
Improved Serum- and Feeder-Free Culture of Mouse Germline Stem Cells
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DOI：
10.1095/biolreprod.114.122317
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发表时间：
2014-10-01
影响因子：
3.6
通讯作者：
Shinohara, Takashi
中科院分区：
文献类型：
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作者：
Kanatsu-Shinohara, Mito;Ogonuki, Narumi;Shinohara, Takashi
精原干细胞（ssc）经历自我更新分裂，这可以在体外重现。为ssc建立无血清培养条件的尝试取得了有限的成功。虽然我们之前报道过在层粘胶蛋白包被板上无血清培养SSC，但由于生长速度和SSC浓度较低，不利于大批量培养SSC。在这项研究中，我们报道了一种新型培养基，可以改善SSC的维持。我们使用Iscove改良的Dulbecco培养基，补充脂质混合物、胎蛋白和敲除血清替代物。在胶质细胞系来源的神经营养因子（GDNF）和成纤维细胞生长因子2 （FGF2）存在的情况下，在层粘连蛋白包被板上培养的ssc可以增殖超过5个月，并在印迹基因中保持正常的核型和雄性激素DNA甲基化模式。生殖细胞移植表明，无血清培养基中的SSCs比添加血清培养基中的SSCs增殖更活跃，两种培养基中SSCs的频率相当。培养的细胞进行种系传播。开发一种新的无血清和无饲料培养SSC的方法将有助于研究微环境对自我更新的影响，并将刺激进一步改进从不同动物物种中获得SSC培养物。
Spermatogonial stem cells (SSCs) undergo self-renewal division, which can be recapitulated in vitro. Attempts to establish serum-free culture conditions for SSCs have met with limited success. Although we previously reported that SSCs can be cultured without serum on laminin-coated plates, the growth rate and SSC concentration were relatively low, which made it inefficient for culturing large numbers of SSCs. In this study, we report on a novel culture medium that showed improved SSC maintenance. We used Iscove modified Dulbecco medium, supplemented with lipid mixture, fetuin, and knockout serum replacement. In the presence of glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), SSCs cultured on laminin-coated plates could proliferate for more than 5 mo and maintained normal karyotype and androgenetic DNA methylation patterns in imprinted genes. Germ cell transplantation showed that SSCs in the serum-free medium proliferated more actively than those in the serum-supplemented medium and that the frequency of SSCs was comparable between the two culture media. Cultured cells underwent germline transmission. Development of a new serum- and feeder-free culture method for SSCs will facilitate studies into the effects of microenvironments on self-renewal and will stimulate further improvements to derive SSC cultures from different animal species.