Recent advances of in vitro culture systems for spermatogonial stem cells in mammals

4.1 Transplant assay of the isolated spermatogonial stem cells

The first transplant assay for the identification of SSCs in mice was performed by Brinster and Zimmermann.24 The recipient mice are depleted for endogenous SSCs through treatment with the anticancer drug, Busulfan, and the transplanted donor-derived SSCs result in complete spermatogenesis. This assay provides functional and quantitative analyses of SSCs, in which donor-derived colonies are generated from single transplanted SSCs.15 In addition, cross-species transplants between mice and rats,25 as well as mice and hamsters,26 results in complete spermatogenesis and the production of healthy offspring. Surprisingly, the transplant of GCs from non-rodent species (Table 1)27-31 (ie, rabbits and dogs32), as well as pigs, cattle, and horses,33 shows the colonization of cells in the mouse testis, but there is a lack of complete spermatogenesis. This finding raises questions regarding whether transplants can be used as a bioassay for the determination of stem cell potential in non-rodent species.34

Apart from the identification of SSCs, a transplant technique has been used for multiple applications, including the restoration of infertility, generation of transgenic and knockout animals, and the evaluation of the culture system and cell markers.35, 36 The transplant of human SSCs into immunodeficient mice was first shown by Nagano, Patrizio, and Brinster.37 The isolated SSCs could colonize and survive for 6 months in mouse testes. The number of SSCs was significantly reduced 2 months after the transplant and no cell differentiation into meiosis was observed. The xenotransplant of human SSCs to the mouse testis by using cultured cells shows a potential regenerative technique for fertility preservation in patients with cancer. Similarly, the autotransplant of SSCs in prepubertal patients with cancer has been considered to be a feasible way to restore infertility after cancer treatment.38

4.2 Biochemical characterization of the spermatogonial stem cells

Defining the SSC populations by using biochemical markers that distinguish them from spermatogonia in other stages of differentiation was a great tool for the isolation of potential SSCs and the development of culture systems in rodents. In recent years, several molecular markers have been identified for SSCs in rodents (Table 2).39-63 Most of these markers are expressed in progenitor SSCs, including As spermatogonia and undifferentiated spermatogonia (Apr and Aal spermatogonia). Traditionally, As spermatogonia have been included in the SSC population that self-renews in order to maintain a foundational stem cell pool and the transition to Apr spermatogonia represents the initial step of spermatogenesis.1, 6 Recent findings show that the SSC population is not limited to the As spermatogonia population.64 Some progenitor SSCs also exhibit stem cell behavior.

Some of these markers are identified as SSC markers in domestic animals (Table 2) and are conserved among mammalian species. The markers, GPR125, GFR1, THY1, ZBTB16, SSEA-4, and PLZF, that have been identified for SSCs in rodents have also been characterized in human spermatogonia and more differentiated GCs.43, 65, 66

5.1 Isolation and enrichment of the spermatogonial stem cells

The isolation and enrichment of SSCs is the first step towards establishing GS cell lines. The isolation of SSCs is challenging because of their limited number in the testis. A two-step enzymatic digestion was first proposed by Davis and Schuetz,67 which is the most widely used technique for the isolation of SSCs in rodents. For further enrichment of SSCs, different approaches, such as differential plating,68 percoll gradient,23 magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) have been used independently or in combination. In livestock species, SSC isolation and enrichment methods have progressed during the last few years. Differential plating is one feasible method for the enrichment of SSCs, along with MACS and FACS for bovine SSCs.69

5.2 Establishment of a culture system for germ cell lines

The limited number of SSCs in the testis4 hampers studies that elucidate biological characteristics and for applying SSCs. One approach to solve this problem is to develop a culture system that supports the self-renewal of SSCs and maintains their GC and stem-cell potentials. Glial cell-derived neurotrophic factor was shown to be the first molecule that regulates the self-renewal and differentiation of mouse SSCs.70 Glial cell-derived neurotrophic factor signals act through the multicomponent receptor complex that is composed of GFRα-1 and RET tyrosine kinases in various cell types.71 The GFRα-1 and RET also have been recognized as spermatogonial markers that are expressed in gonocytes, SSCs, and differentiated spermatogonia.72 These coreceptors of GDNF-mediated signaling have been shown to be necessary for the self-renewal of GCs in rodents.57 Subsequently, Nagano, Ryu, Brinster, Avarbock, and Brinster developed a short-term culture system that is supplemented with GDNF that improves the survival of GCs.15 These cells complete spermatogenesis after transplant into the testis of immunodeficient mice. The long-term culture of SSCs is achieved by adding other growth factors and hormones in addition to GDNF.73 These cells proliferate over a 2 year period (>10⁸⁵-fold) in the presence of GDNF, while maintaining stable genetic and epigenetic properties and restoring spermatogenesis following transplant into the seminiferous tubules of infertile recipient mice. However, the growth factor requirements for the proliferation of GCs is strain-specific: in mice, the C57BL/6 and 129/Sv strains require fibroblast growth factor (FGF) and GDNF,74 while strain DBA requires FGF, GDNF, and epidermal growth factor.75 By using species-specific culture components, culture systems and GC lines have been established in rats,76, 77 hamsters,78 and rabbits.79

Spermatogonial stem cells under appropriate culture conditions acquire embryonic stem (ES) cell-like characteristics called “multipotent GCs,” which were first generated from GCs in the neonatal mouse testis without the introduction of any exogenous reprogramming factor.80 These cell populations failed to form colonies following testicular transplants, which shows that they are devoid of GC potential and have the ability to differentiate into three germ layers. Later, successful evidence of the generation of a multipotent GS cell line was shown for adult mice.60, 81

The successful translation of an in vitro culture of SSCs in rodents led to the establishment of a culture system for human SSCs from prepubertal and adult testes.82, 83 In humans, multipotent stem cell lines have been developed from SSCs by exposing the cells to ES cell culture conditions.84, 85 These cell lines can form a teratoma after they are injected into immunodeficient mice. These findings provide an important foundation for developing methods for the generation of autologous stem cell lines from human SSCs that have been collected from patients with cancer before the initiation of cancer treatment and the subsequent autologous transplant after cancer treatment could be a means for preserving the fertility of male patients with cancer.86

5.3 Spermatogonial stem cell culture in livestock species

In livestock species, long-term culture systems for GCs and the establishment of multipotent GC lines could reduce the time and costs for producing transgenic animals and to preserve endangered species. These systems also could be an alternative for pronuclear microinjection and somatic cell cloning.87 Although several attempts have been made to develop a culture system for livestock species, as shown in Table 3,40, 58, 88-94 most of these studies achieved only short-term SSC cultures. The culture system for bovine SSCs has been demonstrated in the pre-pubertal testis29, 49, 89-91 and the neonatal testis.40 In pigs, cultured SSCs cannot survive more than 1 week50, 92. In these studies, serum was used as an important component in the culture medium for the survival and self-renewal of SSCs. Some undefined factors in the serum induce cell differentiation, whereas others have detrimental effects on ES cells and GC survival in the culture.53, 95 In order to overcome this problem, serum-free culture systems have been developed for long-term cultures of SSCs in mice74, 96 and rats.77 However, no long-term culture system for livestock species has been developed. In the authors’ laboratory, growth factors, matrix substrates for culture dishes, and serum-free supplements have been examined in order to develop a defined system for culturing primitive GCs (gonocytes) from the neonatal bovine testis.58, 97, 98 Poly-L-lysine is a suitable substrate for the selective inhibition of the growth of somatic cells and makes it possible to maintain gonocytes. Among the serum-free supplements that were tested, knockout serum replacement (KSR) in the culture medium supports the proliferation and survival of the gonocytes after sequential passages of the colonies. Under these optimized culture conditions that consist of 15% KSR on poly-L-lysine-coated dishes, the stem cell and GC potentials of cultured gonocytes can be maintained for more than 2 months. Subsequently, also developed was a culture system to maintain the SSCs from immature and adult testis in cattle.99 H The SSCs from the immature testis are cultured under serum-free conditions in the presence of GDNF and bovine leukemia inhibitory factor-conditioning media. Established cell lines resemble ES-like cell properties and express both pluripotent and GC markers. However, the SSCs from the adult bovine testis are cultured in a low-serum concentration media that is supplemented with 6-bromoindirubin-3’-oxime, which is a small-molecule inhibitor of glycogen synthase kinase-3α that leads to the activation of the wingless-type (Wnt)/β-catenin signaling pathway.100 The established cell lines can be maintained under in vitro culture conditions for more than 3 months. This cell line has a normal karyotype and botryoidal morphology that is similar to the male GC lines from mouse SSCs. Taken together, this new finding provides a promising strategy to conserve GCs from livestock species at different stages of animal development.