Background

Over the last several decades, survival rates for childhood cancer have steadily increased. In fact, with the overall cure rate for pediatric malignancies now approaching 80%, current estimates indicate that one in every 530 young adults in the United States will be a survivor of childhood cancer [1]. Unfortunately, many survivors struggle with medical side effects of their treatment including disorders of the endocrine system, cardiac and pulmonary dysfunction, secondary neoplasms, and infertility. Gonadal damage is a relatively common consequence of the treatments used to cure pediatric cancer. The extent of cytotoxic germ cell damage depends on the specific chemotherapeutic agents used and the cumulative doses received. Alkylating agents (particularly cyclophosphamide, ifosfamide, nitrosureas, chlorambucil, melphalan, busulfan, and procarbazine) are the most common class of drugs known to affect gonadal function and their impact has been studied extensively [2]. Additionally, the testes have a very low threshold for radiation exposure, and even small doses are known to be gonadotoxic. As treatment regimens for pediatric oncologic malignancies have improved, more and more survivors are entering their reproductive years [3]. Maintenance of fertility is extremely important with regard to long-term quality of life for these survivors [4, 5]. Consideration must be given to whether a child’s fertility is likely to be impacted by his treatment. Ideally, this should occur before the start of therapy when a window of opportunity may exist to preserve the patient’s future reproductive potential [4–8]. Pubertal males can produce semen prior to starting gonadotoxic therapy and cryopreserve the sperm for future use. Because current methods for oocyte fertilization can utilize as few as one motile sperm (e.g., intracytoplasmic sperm injection [ICSI]), this method has proven to be successful even when the number of cryopreserved sperm is small [5, 9, 10].

Unfortunately, prepubertal males pose a significant challenge for fertility preservation because these boys cannot produce mature spermatozoa for cryopreservation. During embryonic development, primordial germ cells migrate to the genital ridge and differentiate into gonocytes [11, 12]. In the mammalian postnatal testis, gonocytes are the first cell population committed to male germline development. Before puberty, these cells then give rise directly to spermatogonial stem cells (SSCs). In the mouse, which has a short prepubertal period (~3 weeks), some gonocytes transition to SSCs and others undergo an early differentiation directly to type A1 spermatogonia by day 6 of life. During the first 2–3 months after birth in humans, which have a long prepubertal period (~12 years), the gonocytes are replaced by adult dark (Ad) and adult pale (Ap) spermatogonia that are thought to represent the reserve SSC and active SSC pool [12, 13]. These Ad and Ap spermatogonia undergo activation beginning at approximately age 5 years, particularly to type B spermatogonia. By age 10, these SSCs represent about 10% of total spermatogonia. During puberty, the SSCs in all species provide the foundation for spermatogenesis, through self-renewal and differentiation to daughter cells. Although the germ cells of the prepubertal testis contain a small number of the self-renewing SSCs they do not yet have mature spermatozoa. For these at-risk prepubertal boys, current practice does not provide any options for fertility preservation at cancer diagnosis. A potential approach to this issue is the use of cryopreserved testicular tissue. Ideally, prepubertal testicular tissue could be acquired and banked prior to initiating gonadotoxic cancer therapy (Figure 12.1 [14]). Years later, once the patient is ready to begin a family, this tissue could then be thawed and the stored germ cells reimplanted into the patient’s own testes [14, 15]. Alternatively, the number of stored stem cells could be increased by in vitro culture and matured until they can achieve fertilization by use of ICSI [16]. Cryopreservation of the testicular tissue has been shown to be feasible in many species including mouse, rat, pig, baboon, and humans, and the stem cells survive for long periods. Moreover, in several species these cryopreserved stem cells appear to be fully functional and undergo spermatogenesis when returned to the testes of the same species [11, 17, 18]. Unfortunately, the initial testicular biopsy, which is small, contains very few SSCs, and cryopreservation and malignant cell removal results in loss of SSCs; therefore, there is an insufficient number to result in fertility following transplantation. For this transplantation approach to be useful clinically, stem cells from the biopsy sample must be isolated and their number expanded in vitro prior to reintroduction [14]. While significant strides have been made in animal model research in this area, translational use of testicular tissue cryopreservation in humans remains experimental [9, 19].

Figure 12.1 Male germline stem cell preservation. Before treatment for cancer by chemotherapy or irradiation, a boy could undergo a testicular biopsy to recover stem cells. The stem cells could be cryopreserved or, after development of the necessary techniques, could be cultured. After treatment, the stem cells would be transplanted to the patient’s testes for the production of spermatozoa.

From Brinster [14] with permission.

Development of Spermatogonial Transplantation

SSCs are responsible for the continual production of spermatozoa throughout adult life. SSCs and the surrounding cells, the niche, in the seminiferous tubules regulate biological activity of these cells. Considerable research has been dedicated to understanding the interactions between SSCs and the surrounding somatic cells for proper sperm production. For example, Sertoli cells are thought to be extremely important for SSC growth and development by secreting growth factors that regulate these germ cells [20, 21]. Importantly, a critical breakthrough in the characterization of SSCs has been the development of the germ cell transplantation technique [11, 15, 22]. In 1994, Brinster and Avarbock developed the first animal model of SSC transplantation. Injection of spermatogenic cells into the seminiferous tubules gave rise to donor cell-derived foci of spermatogenesis in the recipient testes [22]. Figure 12.2 presents a schematic overview of spermatogonial transplantation in mice. First, transgenic mice carrying a LacZ or green fluorescent protein (GFP) transgene are used as donor mice. The marked donor testis cells are digested to make a single-cell population suspension. The cell suspension is introduced into the testis seminiferous tubules of recipient mice that contain few to no germ cells after treatment with busulfan that destroys endogenous spermatogenesis. The recipient mice can become fertile to father progeny. Notably, resultant offspring carry the donor mouse haplotype [22]. Research utilizing animal models of male infertility (e.g., busulfan treated or nude mice) has demonstrated that there are several methods to use germ cells from testicular tissue to obtain mature spermatozoa for fertilization including autotransplantation, allotransplantation, and xenotransplantation [11, 16, 24]. Autotransplantation is considered more acceptable than allotransplantation or xenotransplantation, although the last two have been used successfully in mouse models, as well as in dog, several species of farm animals and the Macaque [18, 22, 23].

Figure 12.2 Testis cell transplantation method. A single-cell suspension is produced from a fertile donor testis. The cells can be cultured or microinjected into the lumen of seminiferous tubules of an infertile mouse. Only a spermatogonial stem cell can generate a colony of spermatogenesis in the recipient testis. When testis cells carry a reporter transgene that allows the cells to be stained blue, colonies of donor cell-derived spermatogenesis are identified easily in the recipient testes as blue stretches of tubule. Mating the recipient male to a wild-typed female produces progeny, which carry donor genes.

From Brinster [11] with permission.

Morphological Identification of Spermatogonial Stem Cells

The number of SSCs is very low in the testis of an adult mouse. It is estimated that SSCs constitute only about 0.03% of testicular germ cells [25]. Typically, about 10⁶ germ cells are introduced into the recipient testis depending upon stem cell concentration, among which only a few hundred cells could be stem cells. About 20 spermatogenic colonies will develop, depending on the stem cell concentration. Colonization efficiency is estimated to be about 5–10%.

The paucity of SSCs in comparison to differentiating germ cells and somatic cells within the testis has challenged the field to develop reliable markers of these specialized germ cells, so they can be identified unequivocally for subsequent isolation and enrichment in vivo or in vitro. Tangible advances include identification of SSCs or SSC-like spermatogonia by morphology, surface antigen markers, and functional characteristics in relevant animal models. For example, the study of rodent SSCs was previously hampered by the lack of techniques for purification and long-term in vitro maintenance. However, methodological advances have been developed and refined for rodent germ cell identification and transplantation along with improved culture conditions for SSC expansion and growth [21, 26–28]. These techniques have led to the characterization of many aspects of SSC biology, including the identification of growth factors such as glial cell line-derived neurotrophic factor (GDNF) as the main regulator of rodent SSC self-renewal, which is also likely true for most mammalian species [27–29]. Morphologically, gonocytes that give rise to SSCs are larger in diameter than nearby somatic cells, and tend to rest loosely on the basement membrane of the seminiferous tubules. Because of the difference in size and morphological characteristics between gonocytes and somatic cells and absence of the differentiating germ cells, micromanipulation techniques to select these two cell types from single-cell suspensions isolated from prepubertal human and mouse testes have been developed [29]. Selected populations were distinctly homogenous, and virtually pure populations of germ cells and somatic cells have been obtained and validated phenotypically by immunological techniques for well-established markers that differentiate gonocytes/SSCs from somatic cells [29]. For example, selected gonocytes from mouse testes and spermatogonia from prepubertal human testes have been identified using the markers zinc finger and BTB domain containing 16 (ZBTB16), ubiquitin carboxyl–terminal esterase L1 (UCHL1), and deleted in azoospermia-like (DAZL), along with a lack of labeling for GATA binding protein 4 (GATA4), which is found in Sertoli cells but not germ cells [20, 30, 31]. In contrast, selected populations of somatic cells were negative for ZBTB16, UCHL1, and DAZL and positive for GATA4. Data indicate that micromanipulator selection of gonocytes from prepubertal human testis and neonatal mouse testis cell suspensions is an effective technique for the enrichment of germ cells and provides essentially pure (99%) populations of cells for downstream molecular and cellular analyses as well as germ cells for transplantation purposes. These advances also enable future comparisons of SSCs from humans and rodent species for planned transplantation interventions. Moreover, a similarity in self-renewal and survival mechanisms between human and mouse SSCs may exist, because transplantation of testis cells from nonrodent species, including human, into testes of immunodeficient mice allowed the maintenance and limited replication of spermatogonia in the recipient seminiferous tubules for periods of 6–12 months [11, 18, 29]. Additionally, comparison of molecular and cellular fingerprints from isolated human spermatogonia and mouse gonocytes could provide details regarding specific gene expression patterns. The degree and characteristics of gene expression similarity would allow extrapolation of our knowledge about mouse SSCs to the difficult study of human germline cells, and ultimately impact our understanding of human male fertility and infertility.

Isolation, Purification, and Culture of Murine Spermatogonial Stem Cells

As stated earlier, the number of SSCs in the male mouse testes is relatively low, and identification of these cells is not straightforward. Shinohara et al. [32] demonstrated that β₁ and α₆ integrins are specific surface markers for mouse SSCs [32]. Cells that were positive for these markers were selected using a magnetic bead procedure on a testicular cell suspension. Using this method with the transplantation model, there were a greater number of colonies of spermatogenic cells originating from donor cells in the recipient when enhancement of the concentration of the SSCs was used [33]. In 2004, Kubota et al. demonstrated the essential role of GFNF for in vitro proliferation of SSCs, which were enriched from mouse testes. Using a well-established enrichment strategy, (Thy-1+) SSCs were identified and isolated. The stem cells were then cultured in a well-defined serum-free medium, which led to successful expansion, and enabled identification of essential growth factors for this critical cell type. Importantly, Kubota et al. demonstrated that these stem cells grew best in culture with the addition of specific growth factors and their cognate receptors, including GDNF, basic fibroblast growth factor (bFGF), and GDNF family receptor alpha-1 (GFRα1). Kubota and colleagues cultured murine SSCs for 4 months, and then in vivo spermatogenesis was restored after transplantation back into the recipient [27]. Over the last decade, methods have been developed for rodent germ cell transplantation and SSC culture conditions [11, 21, 26–28]. Specifically, glial cell line-derived neurotrophic factor (GDNF) has been established as the main regulator of rodent SSC self-renewal [27, 28]. The c-Ret receptor tyrosine kinase (RET) and the cofactor GDNF-family receptor α1 (GFRα1) bind to initiate intracellular signaling cascades within SSCs [27, 29, 34]. By examining GDNF withdrawal in rodent SSC cultures, several GDNF-dependent genes have been identified, including: B-cell CLL/lymphoma 6, member B (Bcl6b), basic helix-loop-helix family, member e 40 (Bhlhb2), Ets variant gene 5 (Etv5), homeobox C4 (Hoxc4), LIM homeobox 1 (Lhx1), and Tec protein tyrosine kinase (Tec) [29, 34, 35]. Notably, Bcl6b and Etv5 have implicated by several independent research studies to be involved in regulating rodent SSC self-renewal [34–38]. GDNF also activates downstream signaling cascades including phosphatidylinositol 3-kinase (PI3K), serine-threonine kinase AKT family (Akt), and Src family kinase (Src) that impact rodent SSC maintenance and self-renewal. Thus, GDNF is considered a factor critical for SSC self-renewal (Figure 12.3). Importantly, inclusion of GDNF in culture media is essential for SSC self-renewal in vitro, and additional supplementation with bFGF or EGF augments those effects. Currently, SSC culture systems that support long-term SSC self-renewal are available only for mouse, rat, hamster, and rabbit [36, 39]. Additional research is required to determine which specific cell surface and/or intracellular markers are expressed on human testicular SSCs to enable the rapid and reproducible accession of enriched SSC cell populations for downstream analyses and clinical utilization.

Figure 12.3 A proposed model of human spermatogonial stem cell (SSC) self-renewal regulation by glial cell line-derived neurotrophic factor (GDNF), which has been demonstrated to have an essential role in regulating rodent SSC self-renewal. The model is similar to those suggested for mouse SSC self-renewal. In this model, GDNF binds to RET and the GFR_1 coreceptor with possible intracellular protein kinase signaling through SFK and PI3K/AKT downstream pathways to regulate the expression of specific genes, such as Etv5 and Bcl6b, which are involved in SSC self-renewal. However, other genes not regulated by GDNF (e.g., Zbtb16, Taf4b and Lin28), are likely controlled by different signals and may block differentiation but not be involved directly in self-renewal. Genes for these regulatory molecules have been shown to be highly expressed in prepubertal human spermatogonia, mouse gonocytes and mouse SSCs. The basement membrane (green), on which the SSC rests, is generated by the peritubular myoid cells (dark brown) and Sertoli cells (tan).

From Wu et al. [29] with permission.

Culture of Human Spermatogonial Stem Cells

Spermatogenesis in vitro from biopsied germ cells is an excellent alternative for prepubertal boys with malignancies particularly of hematopoietic origin, who carry a risk of relapse after transplantation. The ability to mature stem cells to spermatids in vitro would offer an important option to prepubertal cancer patients. Unfortunately, enormous hurdles remain for bringing the in vitro maturation processes into the clinical setting. Even for autotransplantation of SSCs a major challenge is that human germ cells, like murine germ cells described earlier, will yield a low number of SSCs, as 10⁴ testis cells may contain only two or three stem cells [14, 25]. Methods are needed to isolate and increase the number of human SSCs available to be subsequently autotransplanted or matured in vitro. Unfortunately, the initial biopsy contains very few SSCs, and success of either of these procedures is likely to be difficult. Therefore, for even autotransplantation, a procedure already established in animals, to be used clinically, stem cells from the biopsy sample must be isolated and expanded in vitro prior to reintroduction [14]. SSC isolation from prepubertal human testis biopsy samples by itself is not likely to be sufficient to restore fertility following autologous transplantation because the number of SSCs recovered from a biopsy is small. Our recent study found that testicular biopsies from prepubertal boys (n = 9; range 2–10 years) weighed 31.5 ±3.7 mg and provided 3.9 ± 0.6 × 10⁵ cells per biopsy [29]. The concentration of spermatogonia is predicted to be about 3% of the cell population (estimated to be approximately 11,700 spermatogonia per biopsy), and the number of Ad and Ap spermatogonia with stem cell potential in this population is unknown. For comparative purposes, spermatogenesis restoration to approximately half of the seminiferous tubules of a sterile mutant mouse testis and resultant fertility to approximately half of sterile mice requires transplantation of approximately 150 SSCs per testis. As the adult human testis (about 12 g) is nearly 120 times larger than a mouse testis (about 0.1 g), approximately 18,000 SSCs would need to be transplanted to each human testis for a comparative level of fertility restoration, assuming the same successful response level was obtained to human SSC transplantation [29] . Furthermore, a crucial factor is verifying that the biopsied testis cells do not contain cancerous cells. This process requires use of cell sorting procedures such as fluorescence activated cell sorting (FACS, and see later), effectively necessitating more SSCs than recovered from a biopsy [29]. Therefore, simple transplantation of the SSC cells harvested from a single biopsy is not likely to be sufficient to restore fertility. The culture and expansion in number of healthy, cancer-free SSCs is essential for effective clinical use of human SSC transplantation to restore fertility. A report by Sadri-Ardekani et al. in 2009 suggests that expansion of human germ cells in vitro should be feasible [40]. Research by Bhang et al. published in 2018 offers a novel and promising approach towards the development of culture conditions that support the propagation of SSCs. This was accomplished with testicular endothelial cells. These investigators uncovered that TEC are part of the SSC niche producing GDNF and other factors to support human and mouse SSC in culture [61].