Kubota Hiroshi, Avarbock Mary R, Brinster Ralph L
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA.
Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16489-94. doi: 10.1073/pnas.0407063101. Epub 2004 Nov 1.
Spermatogonial stem cells (SSCs) self-renew and produce large numbers of committed progenitors that are destined to differentiate into spermatozoa throughout life. However, the growth factors essential for self-renewal of SSCs remain unclear. In this study, a serum-free culture system and a transplantation assay for SSCs were used to identify exogenous soluble factors that promote proliferation of SSCs. Mouse pup testis cells were enriched for SSCs by selection with an anti-Thy-1 antibody and cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders in a serum-free defined medium. In the presence of glial cell line-derived neurotrophic factor (GDNF), SSCs from DBA/2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor alpha-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNF-induced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clump-forming cells strongly expressed Oct-4. Under these conditions, SSCs proliferated over 6 months, reconstituted long-term spermatogenesis after transplantation into recipient testes, and restored fertility to infertile recipients. The identification of exogenous factors that allow continuous proliferation of SSCs in vitro establishes the foundation to study the basic biology of SSCs and makes possible germ-line modification by sophisticated technologies. Moreover, the ability to recover, culture indefinitely, and transplant SSCs will make the germ-line of individual males available for periods extending beyond a normal lifetime.
精原干细胞（SSCs）能够自我更新，并产生大量定向祖细胞，这些祖细胞终生都将分化为精子。然而，对于SSCs自我更新所必需的生长因子仍不清楚。在本研究中，利用无血清培养系统和SSCs移植试验来鉴定促进SSCs增殖的外源性可溶性因子。通过用抗Thy-1抗体进行筛选，从小鼠幼崽睾丸细胞中富集SSCs，并将其在无血清限定培养基中的STO（源自SIM小鼠胚胎的硫代鸟嘌呤和哇巴因抗性细胞）饲养层上培养。在胶质细胞源性神经营养因子（GDNF）存在的情况下，来自DBA/2J品系小鼠的SSCs形成了密集堆积的细胞团，并持续增殖。然而，其他品系的小鼠需要添加可溶性GDNF家族受体α-1和碱性成纤维细胞生长因子来支持其增殖。功能性移植试验证明，形成细胞团的细胞确实是SSCs。因此，GDNF诱导的细胞信号传导在SSCs自我更新中起着核心作用。培养中的SSCs数量每5.6天翻倍，且形成细胞团的细胞强烈表达Oct-4。在这些条件下，SSCs增殖超过6个月，移植到受体睾丸后重建长期精子发生，并使不育受体恢复生育能力。鉴定出使SSCs在体外持续增殖的外源性因子，为研究SSCs的基础生物学奠定了基础，并使得通过先进技术进行种系修饰成为可能。此外，恢复、无限期培养和移植SSCs的能力将使个体雄性的种系在超出正常寿命的时间段内可用。
