Laboratory of Cell and Molecular Biology, Department of Animal Science, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan.
Methods Mol Biol. 2020;2155:165-182. doi: 10.1007/978-1-0716-0655-1_14.
Spermatogonial stem cells (SSCs) possess both self-renewal and differentiation abilities to sustain lifelong production of enormous numbers of spermatozoa in males. SSCs hold a unique position among tissue-specific stem cells in adults because of their ability to transmit the genetic information to subsequent generations. Ex vivo expansion of SSCs in conjunction with their transplantation is highly invaluable to study SSCs and develop new reproductive technologies for therapeutic applications. In this chapter, we describe a culture system involving a simple serum-free medium for mouse SSCs. Elimination of the serum from the culture is important to enhance the effects of exogenous factors, which are rather masked by the serum, and to avert the serum-induced inflammatory responses of testicular mesenchymal cells, which cause adverse effects on SSC proliferation. Consequently, using this culture system has proven for the first time that glial cell line-derived neurotrophic factor (GDNF) was found to be the key factor to drive the self-renewing proliferation of SSCs, and fibroblast growth factor 2 enhanced the GDNF-dependent proliferation of SSCs. Besides determining these two key cytokines, the simplicity of the system enabled individual modification of its components to develop long-term cultures of rat and rabbit SSCs. The basics of these culture systems will enable development of the culture conditions for human and other mammalian SSCs in the near future.
精原干细胞（SSCs）具有自我更新和分化能力，能够在男性中维持大量精子的终生产生。SSCs 在成人组织特异性干细胞中具有独特的地位，因为它们能够将遗传信息传递给后代。SSCs 的体外扩增与移植结合在一起，对于研究 SSCs 和开发新的治疗性生殖技术具有极高的价值。在本章中，我们描述了一种涉及简单无血清培养基的小鼠 SSCs 培养系统。从培养物中去除血清对于增强外源性因素的作用非常重要，因为血清会掩盖外源性因素的作用，并且可以避免血清诱导的睾丸间质细胞炎症反应，这种反应会对 SSC 的增殖产生不利影响。因此，使用这种培养系统首次证明，胶质细胞系衍生的神经营养因子（GDNF）被发现是驱动 SSC 自我更新增殖的关键因素，而碱性成纤维细胞生长因子 2 增强了 GDNF 依赖的 SSC 增殖。除了确定这两种关键细胞因子外，该系统的简单性还使得可以对其成分进行单独修饰，从而开发出大鼠和兔 SSCs 的长期培养。这些培养系统的基础将使人们能够在不久的将来开发人类和其他哺乳动物 SSCs 的培养条件。
