Rapid expansion of the spermatogonial stem cell tool box.

R ecently, great technological progress has been achieved in spermatogonial stem cell (SSC) research. In this issue of PNAS, Kanatsu-Shinohara et al. (1) describe yet another important novel use for SSCs. Based on a successful long-term culture protocol for mouse SSCs, this group has designed a way to produce knockout mice from SSCs with an efficiency that is at least comparable with that of embryonic stem (ES) cell-based methods. SSCs were transfected by applying methods used for ES cells and transplanted into recipient mouse testes to produce sperm carrying the desired mutation. This procedure may enable the efficient production of transgenic animals in species from which no ES cells can be made as yet. Before 1994, spermatogonial stem cell numbers could be assessed only by cell counts (2, 3). Then Brinster and colleagues introduced a functional assay for SSCs, the SSC transplantation technique (4, 5). This method has greatly boosted research on SSCs. However, despite efforts by many groups, it remained problematic to culture SSCs and propagate these cells in vitro, hence limiting SSC availability. The breakthrough came when Kanatsu-Shinohara et al. (6) succeeded in culturing SSCs for at least 5 months, achieving a 1014-fold increase in SSC numbers [called germ-line stem (GS) cells by the authors]. These cultured SSCs remained capable of colonizing recipient mouse testes upon transplantation, giving rise to normal spermatogenesis (6). SSCs could be cultured either without serum or without a feeder layer (7), remained genetically and epigenetically intact (8), and could be cultured also in an anchorage-independent way (9). The culture period could be extended to at least 2 years, and a 1085fold increase in SSC numbers was achieved in this way (8). The factors leading to this breakthrough in culture possibilities probably lay in the use of a proprietary culture medium of unknown composition and a combination of added growth factors, including glial cell line-derived neurotrophic factor (GDNF) (6). Very large numbers of genetically normal and transplantable mouse SSCs now can be produced in vitro and used as a reliable starting material to make transgenic animals (Fig. 1). The starting material in the culture experiments was germ cells from newborn mice. In mice, spermatogenesis starts shortly after birth, and the only germ cells present at that time are early differentiating spermatogonia and SSCs (2, 12, 13) (Fig. 1). Therefore, the germ cells already were enriched for SSCs in comparison with the normal adult testis. Interestingly, after 4–7 weeks in culture, colonies of ES-like cells were formed, called mGS cells (10). These mGS cells were multipotential and able to form various types of somatic cells in vitro just like ES cells. The results indicated that the mGS cells were formed by the cultured GS cells themselves at a low frequency and were not some leftover, earlier type of germ cells still present at birth. The formation of ES-like cells by the GS cells may depend on the age of the mice from which the population of SSCs was isolated initially. KanatsuShinohara et al. (10) did not find ESlike cell formation when testes of 4to 8-week-old WT mice were used to isolate SSCs. This result could point to a differentiation step of SSC shortly after birth, preventing the formation of ESlike cells in culture. However, recently, Guan et al. (11), using a different culture protocol, found multipotent ES-like cell formation, called maGSCs by the authors, from cultured spermatogonia isolated from 4to 6-week-old mice. In addition, Kanatsu-Shinohara et al. (10) found ES-like cell formation from germ cells isolated from 3to 8-weekold p53 knockout mice instead of WT mice. Taken together, it seems possible that the transition from SSCs to ESlike cells still can be made in older mice. Further studies are needed to find out whether there is a maximum age of the donor mice, and ES-like cell formation from SSCs also should be studied in other mammals, including humans. This amazingly fast development in the SSC field now paves the way for important scientific and technological applications for SSCs. First, the propagation of stem cells achieved in the mouse (1085-fold increase) will encourage attempts to in vitro propagate SSCs from other mammals, including humans. Positive results already have been obtained in the rat (14), and we