Techniques for Culturing Spermatogonial Stem Cells Continue to Improve

In this issue of Biology of Reproduction, Kanatsu-Shinohara et al. [1] report a novel technique for culturing spermatogonial stem cells (SSCs) in the absence of both serum and feeder cells. This achievement has significant implications in SSC research and applications. To appreciate this study, it is useful to briefly review the past progress in the development of SSC culture systems. The study of SSCs dates back to the 1950s [2]; however, functional analyses of SSCs were limited until 1994, when the group led by Ralph Brinster demonstrated the regeneration of complete spermatogenesis following transplantation of donor mouse testis cells into recipient testes [3, 4]. Because stem cells are defined by their ability to reconstitute an adult tissue [5], this transplantation technique provided an unequivocal detection method of SSC potential. Nonetheless, the technique alone did not allow for the experimental manipulation of SSCs or for the dissection of their cellular biology. The SSC culture was therefore expected to become an important tool in understanding the mechanisms that control SSC survival, self-renewal, proliferation, and differentiation. The first SSC culture was reported in 1998 by the Brinster laboratory, using the mouse as a model [6]. Although it was a crude system in which entire seminiferous tubule cells were cultured in a medium containing fetal bovine serum (FBS), the study showed that long-term maintenance of SSCs in vitro was possible if embryonic fibroblasts were provided as feeder cells. The development of SSC enrichment techniques [7, 8] improved the efficacy of SSC culture [9, 10]. These studies also showed that pup SSCs divided more actively than adult SSCs [9, 11]. The discovery of glial cell line-derived neurotrophic factor (GDNF) as a critical factor for SSC self-renewal was pivotal to the development of SSC culture. Using transgenic mice, Meng et al. [12] demonstrated that overexpression of GDNF led to accumulation of undifferentiated spermatogonia, whereas its haploinsufficiency resulted in the loss of stem cell reserve. On this basis, two research groups, led by Takashi Shinohara [13] and by Ralph Brinster [14], established mouse SSC culture systems. In both, SSCs self-renewed virtually indefinitely and proliferated robustly in the presence of GDNF and FGF2. Additional elements common to both techniques were reduction of testicular somatic cells and enrichment of SSCs and the use of embryonic fibroblasts as feeder cells. The Shinohara method used a low level (1%) of FBS, whereas the Brinster method employed a serum-free medium with its defined chemical components, including a mixture of free fatty acids [15]. The Shinohara group further improved its culture conditions to the point of development of a system where either feeder cells or serum was omitted [16]. Following from these advances, they now present an SSC culture that contains neither feeder cells nor serum [1]. To achieve this, they used laminin as a culture substrate and supplemented a serum-free medium with fetuin and lipid-associated components. Fetuin, a major protein in FBS, enhanced cell attachment to laminin but was not sufficient for sustained cell proliferation. The authors resolved this problem by adding lipid-associated components to the medium, suggesting significant roles for lipids in promoting the activity of SSCs and their progeny. With these results, it is reasonable to attribute the success of the Brinster method partly to lipid inclusion in the medium. The elimination of feeder cells and serum is important, because these components introduce unknown factors into a culture environment. Therefore, the current study represents a significant improvement of the SSC culture technique. On the other hand, it also indicates issues remaining to be addressed for establishing a defined SSC culture system. Here, three of these issues are considered. First is the culture medium. The medium used in the current study is based on StemPro medium, a proprietary product that might include undisclosed components that are detrimental or beneficial to SSCs. The medium is also complex, containing various growth factors, such as epidermal growth factor and leukemia-inhibitory factor as well as steroid hormones. Lipid components used in this study are commercially produced from the serum, and a batch-to-batch difference in the products may affect SSC behavior. Hence, a better-defined medium needs to be established. A similar issue also applies to the Brinster method. Although their medium is simpler and completely defined, feeder-free culture has not been reported using this medium. Further improvement is thus necessary, and identification of feeder cell function could be valuable. The second issue is serum. Culture techniques have been developed for various types of stem cells, and a serum-free condition is common for nearly all of the techniques. Does this imply that serum is universally detrimental to stem cells in vitro? The current study indicates that serum does have positive effects on SSCs in culture, and notably shows that the proportion of SSCs in cultured cells was lower than that found in the presence of serum. Apparently, there are still unknown serum components that may improve the SSC culture. Correspondence: FAX: 514 843 1662; e-mail: makoto.nagano@muhc.mcgill.ca

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