Zhang Pengfei, Chen Xiaoxu, Zheng Yi, Zhu Jinshen, Qin Yuwei, Lv Yinghua, Zeng Wenxian
1 College of Animal Science and Technology, Northwest A&F University , Shaanxi, China .
2 Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam , Amsterdam, the Netherlands .
Stem Cells Dev. 2017 Aug 1;26(15):1121-1131. doi: 10.1089/scd.2017.0018. Epub 2017 May 4.
Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.
精原干细胞（SSCs）为雄性成年后的精子发生和生育能力奠定了基础。将精原干细胞的基因操作与生殖细胞移植相结合，为基因治疗和转基因动物的生产提供了一种新方法。然而，哺乳动物睾丸中精原干细胞的稀少仍然是相关应用的一个障碍，这使得精原干细胞的体外扩增成为一个先决条件。尽管如此，大型动物精原干细胞的长期培养系统尚未建立。在本研究中，我们为猪未分化精原细胞开发了一种优化的体外培养条件。通过优化的差异种植从7日龄猪睾丸中分离并富集生殖细胞。我们测试了不同的饲养层，发现新生自体支持细胞比SIM小鼠胚胎来源的硫鸟嘌呤和哇巴因抗性（STO）细胞系及成年支持细胞表现更好。还研究了几种生长因子的作用。使用新生支持细胞作为饲养层，并在杜氏改良 Eagle 培养基：营养混合物 F-12（DMEM/F12）培养基中添加 10% KSR 和四种细胞因子，未分化的精原细胞可以在体外增殖至少2个月而不失干性。精原干细胞标志物的表达表明培养的细胞保持了精原干细胞的表达谱。此外，异种移植和体外诱导表明，长期培养的细胞分别保留了在体内定植和在体外分化的能力，证明培养的细胞中存在精原干细胞。总之，本研究中描述的条件可以支持猪未分化精原细胞的正常增殖，保持干性和正常核型至少2个月。这种培养系统将作为未来研究的基本改进，并促进精原干细胞生物学和雄性生殖细胞基因操作的研究。
