Biology and manipulation technologies of male germline stem cells in mammals

Abstract Background Spermatogonial stem cells (SSCs) are the origin of sperm and defined by their functions of “colonization in the testis” and “spermatogenesis”. In vitro manipulation techniques of SSCs contribute to a wide variety of fields including reproductive medicine and molecular breeding. This review presents the recent progress of the biology and manipulation technologies of SSCs. Methods Research articles regarding SSC biology and technologies were collected and summarized. Main findings Dr. Ralph Brinster developed the spermatogonial transplantation technique that enables SSC detection by functional markers. Using this technique, cultured SSCs, termed germline stem (GS) cells, were established from the mouse. GS cells provide the opportunity to produce genome‐edited animals without using zygotes. In vitro spermatogenesis allows production of haploid germ cells from GS cells without spermatogonial transplantation. The recent advancement of pluripotent stem cell culture techniques has also achieved production of functional GS‐like cells in addition to male/female germ cells. Conclusion Although in vitro manipulation techniques of GS cells have been developed for the mouse, it appears to be difficult to apply these techniques to other species. Understanding and control of interspecies barriers are required to extend this technology to nonrodent mammals.


| DEFINITI ON AND DE TEC TI ON OF SSC
Stem cells are defined by two particular functions: self-renewal and differentiation. 1 For example, hematopoietic stem cells (HSCs) can produce multipotent progenitor cells that proliferate rapidly to generate more committed progenitor cells, such as common lymphoid and myeloid progenitor cells, toward producing the various types of mature effector cells. 2 A HSC can be detected by a transplantation assay in which the single cell produces T-lymphoid cells, B-lymphoid cells, and myeloid lineage cells to be identified as a HSC. 3,4 Somatic stem cells cannot be recognized and isolated precisely by a prospective approach such as fluorescence-activated cell sorting (FACS).
Hence, the stem cell transplantation assay is the only retrospective method to detect and count the number of stem cells based on their functional definition.
Spermatogonial stem cells are the only cell type that can produce differentiating progeny for sperm production. Therefore, SSCs are categorized as unipotent stem cells. The transplantation assay for SSCs was primarily developed by Brinster and Zimmermann. 5 In their study, a testis cell suspension containing SSCs was injected into a seminiferous tubule of an infertile mouse testis (eg, busulfantreated mice and congenitally infertile Kit W /Kit W-v mice). The transplanted SSCs colonized the recipient seminiferous tubule and started spermatogenesis. The generated sperms were able to produce offspring, indicating that the colonized cells were SSCs. 6

SSC injection
can be performed via the efferent duct and/or rete testis ( Figure 1). 7 Subsequent studies have demonstrated that one colony generated by spermatogonial transplantation is derived from a single SSC, 8,9 demonstrating that the spermatogonial transplantation assay can be used for SSC quantitation.
This technique led to the possibility of in vitro SSC manipulation. The primary application was developed by Nagano et al who infected SSCs in vitro with a retroviral vector carrying a LacZ transgene, which colonized infertile mice. 10,11 This study demonstrated the possibility of in vitro SSC manipulation. However, simultaneously, it was strongly suggested that the SSC culture system is beneficial for further advancement of SSC manipulation.

| S ELF-RENE WAL FAC TOR S FOR SSC S AND E S TAB LIS HMENT OF G ERMLINE S TEM (G S) CELL S
Maintenance and expansion of SSCs are supported by several soluble factors. Thus far, multiple cytokines, such as colony stimulating factor 1 (CSF1), wingless-type MMTV integration site family (WNT) 5A, WNT3A, vascular endothelial cell growth factor A, fibroblast growth factor (FGF) 8, and WNT6, are reported to be a functional in SSC maintenance and expansion. [12][13][14][15][16][17][18] Among these cytokines, glial cell line-derived neurotrophic factor (GDNF) is the primary factor that is indispensable for SSCs. Meng et al reported that haploinsufficiency of Gdnf results in gradual loss of spermatogenesis, whereas Gdnf overexpression causes hyperproliferation of undifferentiated spermatogonia. 19 Mutation in the Ret proto-oncogene also resulted in a similar phenotype of spermatogonia. 20,21 Discovery of GDNF allowed establishment of SSC lines. The first report of in vitro SSC culture was published by Nagano et al, in which testis cells were cultured on mitomycin-treated STO feeder cells with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Although the testis cells maintained SSC activity even after 111 days of culture in the best case, obvious expansion of SSCs was not observed. 22 Long-term culture and expansion of SSCs in vitro were achieved by Kanatsu-Shinohara et al. using epidermal growth factor (EGF), leukemia inhibitory factor (LIF), FGF2, GDNF, and mitomycin C-treated mouse embryonic fibroblasts as feeder cells. 23 In their culture system, testis cells derived from a pup of the DBA/2 strain formed grape-like clumps of cells and proliferated for more than 4 months in a logarithmic manner without losing colonization activity in testes of infertile mice. Moreover, haploid male germ cells could produce offspring, demonstrating that the cultured cells possessed the proper SSC activity. Hence, these cells were named GS cells ( Figure 2).
Subsequently, some studies reported comparable results regarding GS cell derivation from other mouse strains under similar conditions. 24,25 These results suggested that the combination of mouse strain and age, feeder cells used, and serum concentration affected the in vitro expansion of SSCs.
FGF2 was thought to be a supportive factor for GS cells.
However, we found that GS cells can be expanded with GDNF or FGF2 alone in vitro. This finding suggested that GDNF is dispensable for SSC maintenance and self-renewal. 26 Intriguingly, FGF2-cultured spermatogonia have a morphology, doubling time, and SSC activity distinct from those of GDNF-cultured spermatogonia. In addition, the former cells survive and proliferate without MAP2K1 (dual specificity mitogen-activated protein kinase 1) activation, whereas the latter cells require its activation, suggesting that FGF2 promotes survival and proliferation of SSCs F I G U R E 1 Transplantation of SSCs via the efferent duct. In this procedure, a glass capillary is inserted into the rete testis via the efferent duct. This photo demonstrates injection of a trypan blue solution into seminiferous tubules, instead of SSCs/GS cells. The image was obtained from a previous review with permission from the Japanese Journal of Embryo Transfer 129 via signals distinct from GDNF signals. In fact, we found that FGF2-stimulated undifferentiated spermatogonia form colonies with characteristics distinct from those of GDNF-induced undifferentiated spermatogonia in vivo. 27 In this study, forced input of strong FGF2/GDNF signals was applied directly in mouse testes using intelligent biomaterials. Although we found that both factors induced hyperproliferation of GDNF family receptor alpha-1 (GFRA1)-expressing undifferentiated spermatogonia, FGF2induced GFRA1 + spermatogonial colonies exhibited a morphology distinct from those induced by GDNF ( Figure 3). In addition, we found that FGF2-induced spermatogonia were prone to express retinoic acid receptor γ (RARG). Considering that expression of RARG is sufficient for retinoic acid-mediated differentiation of undifferentiated spermatogonia into differentiating spermatogonia, 28 FGF2 is quite unique because this molecule contributes to spermatogonial differentiation despite the fact that it is a bona fide self-renewal factor for SSCs. These observations raise the possibility that FGF2 plays a role distinct from GDNF in controlling the fate of SSCs/undifferentiated spermatogonia in vivo.
Our most recent studies revealed expression of Fgf2 in the germ cell population, while Gdnf is expressed in Sertoli cells and peritubular myoid cells. 27,[29][30][31] In these studies, germ cell depletion increased the relative expression of Gdnf, while Fgf2 was relatively suppressed. 27,29 Considering that germ cell-depleted conditions are relatively appropriate for expansion rather than differentiation of transplanted undifferentiated spermatogonia/SSCs, 32,33 the Gdnf/Fgf2 ratio might affect fate determination of undifferentiated spermatogonia/SSCs ( Figure 3).
Although our recent studies demonstrate that FGF2 acts as a differentiation factor by expanding the RARG + subset of undifferentiated spermatogonia, FGF2 is still a promising factor to expand SSCs in vitro. Indeed, FGF2-cultured spermatogonia possess considerable SSC activity even after in vitro culture for more than 4 months under GDNF-free conditions. 26 Moreover, it has been demonstrated that FGF2 supports GDNF-mediated expansion of undifferentiated spermatogonia in vitro. 23 Although CSF1 and WNT5A were confirmed to support expansion of an SSC subset in vitro, 12,13,18 it should be determined whether the other candidate factors mentioned above can support GS cell proliferation.

F I G U R E 2 Morphology of mouse GS cells. GS cells form grape-
like cellular clusters on a feeder layer of mitomycin C-treated mouse embryonic fibroblasts in the presence of GDNF and FGF2. Scale bar = 100 μm F I G U R E 3 Functional differences between FGF2 and GDNF in the testicular microenvironment. FGF2-induced GFRA1 + spermatogonia are a differentiation-prone subset because these cells tend to express RARG, the receptor for retinoic acid that induces spermatogonial differentiation. However, GDNF-induced GFRA1 + spermatogonia tend to be negative for RARG, suggesting that these cells are a differentiation-resistant subset. 27 The Gdnf/Fgf2 ratio decreases along with postnatal testicular development and increases during regeneration. 29 During testicular development or regeneration (eg, after busulfan-mediated germ cell depletion), Gdnf expression upregulates in the testis, while undifferentiated spermatogonia expand their population without differentiation. 29 34 Additionally, these cells are susceptible to drug selection. 35 Therefore, GS cells are considered to be more suitable than ES cells for genome editing of germline lineages.
Using this property, transgenic/knockout mice have been produced via lipofection, electroporation, and retroviral vector infection of GS cells. 35,36 In addition, lentivirus-, adenovirus-, and adeno-associated virus-mediated gene transductions are suitable for GS cells. [37][38][39][40] Moreover, GS cell-mediated transfer of an ectopic chromosome was achieved by Shinohara et al. 41 Since editing of the animal genome has become more common with the application of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 technology to mammalian zygotes, [42][43][44] GS cells have also been demonstrated to be an alternative platform for CRISPR-Cas9-mediated genome editing. 45,46 Zygote-mediated genome editing is superior to produce genome-edited animals in a short period. However, the editing accuracy cannot be verified until production of the offspring. Although accuracy verification of ES cell-mediated genome editing can be performed before producing genome-edited offspring, 47,48 GS cell-mediated genome editing is thought to be more superior because of its genomic stability. 34

| IN VITRO S PERMATOG ENE S IS OF G S CELL S
Although GS cells primarily produce mature sperm by spermatogo-  60 Simultaneously, they also explored cytokines essential for in vitro induction of the germ cell fate in primary epiblast tissue. 61 By combining these achievements, they succeeded to induce functional male germ cells from ES and iPS cells. 62 In these reports, they pri-

| APPLI C ATI ON OF G S CELL TECHNOLOG IE S TO NONRODENT S PECIE S
As described above, in vitro manipulation techniques for GS cells, including transplantation, in vitro expansion, genome editing, and sperm production, were established in the mouse. These techniques can contribute to the fields of biological science, agriculture, and medicine. Therefore, it is required to apply these techniques to nonrodent mammals. However, there are some remaining issues to be resolved as discussed below.

| Functional assessment of GS cells
Spermatogonial transplantation is critical for SSC manipulation.

Establishment of GS cells was achieved only in rodents including
the mouse, rat, hamster, and rabbit. 23,[82][83][84][85][86] Since establishment of mouse GS cells, many trials have been reported to establish GS cells from other mammalian species by following the mouse GS cell culture method in combination with spermatogonial transplantation.
However, SSCs derived from bovines, pigs, and humans, have not been expanded in vitro. 72,73,[87][88][89][90][91] In the most recent report, Oatley et al developed a culture condition for cattle undifferentiated spermatogonia to form germ cell colonies with a grape-like morphology that resembles mouse GS cell morphology. 92 Although obvious expansion of GS cell-like cells was not observed, their study suggested that the choice of basal medium, cultivation temperature, and species matching between SSCs and feeder cells for culture might be essential to establish stable GS cell lines from testes of mammals other than rodents. To establish a universal culture method for mammalian GS cells, a serum-and feeder-free culture system might be valuable. The first report regarding successful GS cell culture under serum-free conditions was published by Kubota et al. 25 Moreover, a feeder-free culture system was primarily established by Kanatsu-Shinohara et al. 93 They also succeeded to expand GS cells in suspension culture. 94 As a combined approach, a serum-and feeder-free long-term culture system was established in 2011. 95 Furthermore, they eliminated the chemically undefined supplements from the serum-and feeder-free culture system. 96 In the case of pluripotent stem cells, chemically defined culture systems contribute to maintaining the ground state of pluripotency under which pluripotent stem cells can highly contribute to forming chimeric offspring after injection into a blastocyst. 97,98 This concept was expanded to human pluripotent stem cells. 99 Improvement of chemically defined culture for GS cells will contribute to not only establishment of animal/human GS cells, but also understanding the self-renewal mechanism of SSCs.
In addition to the culture conditions, several putative factors are considered to hamper the establishment of animal/human GS cells. First, it must be noted that testicular somatic cells from large animals proliferate more rapidly than those from rodents. In the case of mice, the majority of testicular somatic cells can be excluded by differential plating with residual cells overwhelmed by proliferating SSCs. 23 This procedure was thought to be applicable to large mammals. 90,92 However, testis somatic cells from piglets show a much higher proliferation activity than those from mouse pups. In my experience, piglet somatic cells overwhelm SSCs in vitro in the present culture condition even after differential plat- Aldefluor reagent and CDy1 have been reported to be effective for SSC purification. The former visualizes the cellular activity of aldehyde dehydrogenase (ALDH). In fact, mouse SSCs are found within the ALDH − subset of CD9 + or CDH1 + testis cells in mice. 113 The latter dye positively stains an SSC-enriched subset of both testicular and GS cells. 114 Recent reports have also demonstrated that the magnitude of Tert expression coincides with cell surface expression of GFRA1, suggesting that TERT expression/activity can be a marker for mouse SSCs. 115,116 Although it is interesting to apply these techniques to purify SSCs from nonrodent animals, there is a concern regarding quantification of the SSC number in the purified subset. Therefore, improvement of the SSC transplantation assay should be pursued simultaneously.

| In vitro spermatogenesis from GS cells
In vitro spermatogenesis to produce animal and human sperm is one of the topics in germ cell biology and its application. However, this technique is still limited to the mouse, probably because of the interspecies barrier described above. In particular, it should be noted that the volume of mesodermal tissue around the seminiferous tubule in the rodent testis is quite smaller than that in other mammals.
Considering the efficient exchange of gas and medium in the organ culture system, testis tissue of rodents might be exceptionally suitable for the present technology. Furthermore, Kanatsu-Shinohara et al. succeeded in partial reconstitution of SSC-Sertoli cell interactions for long-term maintenance of SSC activity in vitro. 128 Improvement of this culture system in combination with screening of small chemicals that facilitate spermatogenesis will contribute to develop in vitro spermatogenesis technique without using an organ culture system. Science Foundation, and Inamori Foundation.

Conflict of interest:
The author declares no conflict of interest.
Human rights statement and informed consent: In this review, the author did not conduct any experiments using human-derived materials. Animal studies: In this review, the author did not conduct any animal experiments.