Martin Laura A, Seandel Marco
Department of Surgery, Weill Cornell Medical College, NY, USA.
J Vis Exp. 2013 Feb 25(72):e50017. doi: 10.3791/50017.
Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells(1-3). Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines(4). However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state. Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors(5,6). Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies(7). Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types(8). Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC(2,3). The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)(3). Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.
睾丸中的精原干细胞（SSCs）是成年哺乳动物干细胞的经典例子，能在动物几乎整个生命周期内维持生育能力。虽然体内调控自我更新和分化的确切机制很难研究，但此前已开发出多种系统，通过结合使用特殊培养基和饲养细胞在体外扩增小鼠SSCs（1-3）。此前大多数对SSCs生物学的体外研究都从新生小鼠中获取细胞系，这可能是因为获取成年细胞系存在困难（4）。然而，在大多数小鼠品系中，睾丸一直要到约5周龄才发育成熟。在出生后的早期阶段，睾丸的结构以及体细胞和生精细胞的生物学特性都会发生显著变化，包括众多干细胞相关基因表达水平的改变。因此，源自新生小鼠的SSC系可能无法完全重现成年睾丸达到稳态后持续存在的成年SSCs的生物学特性。历史上有几个因素阻碍了成年SSC系的产生。首先，由于内在或外在因素（5,6），成年期功能性干细胞的比例可能会下降。此外，与其他成年干细胞一样，不使用免疫筛选或其他分选策略的组合，很难从成年睾丸总细胞中充分富集SSCs（7）。常用策略包括使用隐睾小鼠作为供体细胞来源，因为其干细胞与其他细胞类型的比例更高（8）。基于从初始培养物中去除体细胞会破坏与干细胞生态位的相互作用这一假设，而这种相互作用对SSC存活至关重要，我们此前开发了一些方法来获得成年细胞系，这些方法不需要免疫筛选或隐睾供体，而是采用在培养物中对SSCs进行连续富集，以下简称SESC（2,3）。下面描述的方法涉及一个简单的程序，即通过解离成年供体的生精小管来获得成年SSC系，然后将细胞接种到由睾丸基质细胞系（JK1）组成的饲养层上（3）。通过连续传代，培养物中强烈贴壁的污染性非生殖细胞会逐渐减少，同时SSCs得以富集。以这种方式产生的培养物包含处于不同分化阶段的精原细胞混合物，基于长期自我更新能力，其中含有SSCs。SESC方法的关键在于，它能使SSCs在截然不同的微环境中，从体内自我更新艰难过渡到体外长期自我更新，产生不含污染性体细胞的长期SSC系，从而便于后续对SSCs进行实验操作。
