Germline stem cells: Towards the regeneration of spermatogenesis

Abstract

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or pre-reproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg or embryo banking, followed by in vitro fertilization and/or embryo transfer. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

Introduction

High dose chemotherapy, whole body radiation or radiation to the gonads can cause permanent infertility (1). This is a significant human health concern because over 75,000 people under the age of 40 in the United States are diagnosed with cancer each year and most are cured (2). Thus, cancer patients can look beyond their diagnosis and treatment to quality of life after cancer. Parenthood is important to cancer survivors and distress over infertility can have long-term psychological and relationship implications (3). Therefore, the American Society for Clinical Oncology (ASCO) (4) and the American Society for Reproductive Medicine (ASRM) Ethics Committee (5) recommend that the reproductive risks of gonadotoxic therapies and options for preserving fertility be discussed with patients before initiating treatment. While adoption and third-party reproduction provide alternative family building options, the available data indicate that most cancer survivors prefer to have their own biological children (4).

Post-pubertal adolescent and adult males have the option to cryopreserve sperm prior to oncologic treatment (Figure 1, Top). This is a simple and established method for preserving fertile potential and allows men to father their own genetic children. Nearly 17,000 men between the ages of 15 and 44 are diagnosed with cancer each year in the United States and nearly 2385 survivors will receive a treatment that puts them at high risk of azoospermia (SEER 2010)(2, 6). Unfortunately, only about 24% of men in this age range cryopreserved semen prior to their oncologic treatment (7). Therefore, we calculate that each year in the United States, over 1800 adult cancer survivors will be infertile with azoospermia and have limited options to have their own biological children because they did not save a semen sample. In some cases, sperm can be recovered surgically from small focal areas of spermatogenesis in the testes using the testicular sperm extraction (TESE) method and used to fertilize oocytes by intracytoplasmic sperm injection (ICSI)(8).

Figure 1.

Standard and experimental options for preserving male fertility. Top, sperm obtained by ejaculation or surgical retrieval from the testes or epididymides are competent to fertilize oocytes using assisted reproductive techniques including intrauterine insemination (IUI), in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (ICSI)) that are standard in most fertility clinics. These options are not available to prepubertal boys who are not producing sperm or to adult azoospermic men. Bottom, testis tissue obtained via biopsy from prepubertal boys contains SSCs that can produce sperm in the context of the intact tissue by xenotransplant, organ culture or autologous transplantation back into the individual (orange boxes). Sperm retrieved from cultured or transplanted tissue can be used for ICSI. Cells in suspension obtained from biopsied testicular tissue can be transplanted back into the endogenous seminiferous tubules of the patient (blue boxes). SSCs in the suspension can regenerate spermatogenesis and, in some cases, fertility. For infertile individuals who did not preserve germs cells before gonadotoxic therapy, induced pluripotent stem cells (IPSCs) may be produced from his somatic cells (e.g., skin or blood) to differentiate into transplantable germ cells (PGCs or SSCs) or haploid germ cells that can be used for ICSI (red boxes). Excerpted with permission from Clark AT, Phillips BT and Orwig KE 2011 NATURE MEDICINE 17:1564–1565.

There are no options to preserve the fertility of prepubertal boys, who are not yet making sperm. This is a significant problem because about 5131 boys under the age of 15 in the United States are expected to develop cancer each year and 83% are expected to survive (SEER, 2010)(2). A report from the Childhood Cancer Survivor Study indicates that the cytotoxic therapies for cancer reduce the number subsequently able to have children by 44% (6, 9). Based on these statistics, we calculate that each year in the United States, 1874 young male cancer patients will become sterile due to their treatment. In addition to cancer survivors, over 500 patients under the age of 20 receive hematopoietic stem cell (HSC) transplants each year in the United States for non-malignant conditions (e.g., bone marrow failure, blood and immune deficiencies, autoimmune disorders)(10). Myeloablative conditioning therapy prior to bone marrow transplantation is associated with a high risk of infertility (4, 9, 11, 12). The ASCO report notes that “Impaired future fertility is difficult for children to understand, but potentially traumatic to them as adults” (4). The available data indicate that greater than 80% of parents consented to fertility preservation procedures on behalf of their children prior to initiation of gonadotoxic therapies (13, 14).

The summed incidence of chemotherapy or radiation-induced male infertility that cannot be treated with existing reproductive therapies is approximately 4000 individuals each year in the United States. Therefore, responsible development of novel therapies to help these patients have biological children has a significant potential impact.

Promising results in animal models and human cell lines (Figure 1, Bottom) have generated enthusiasm that stem cells might be used or manipulated to preserve and/or restore the fertility of patients who are not producing sperm and have no other options to protect their future fertility before receiving gonadotoxic chemotherapy or radiation treatments (14–34). We will review the methods of spermatogonial stem cell transplantation (Figure 1, blue boxes), testicular tissue grafting, testicular tissue organ culture (Figure 1, orange boxes) and induced pluripotent stem cell differentiation into gametes or transplantable male germline stem cells (Figure 1, red boxes). Table 1 summarizes published reports detailing the progress of each method. Enthusiasm for these experimental stem cell technologies is tempered by concerns about feasibility and safety, particularly for the vulnerable prepubertal patient population (14, 35–37). Questions about feasibility stem from the early stage of technology development, uncertainty about optimal freezing conditions, concerns that small testicular biopsy will contain few stem cells and the lack of culture conditions to expand human germline stem cells. Questions about safety are associated with the risks of surgery to obtain testicular tissue and the potential for malignant contamination in transplanted cells or tissue. Nonetheless, academic centers around the world are already cryopreserving testicular tissues for prepubertal boys and men in anticipation that stem cells in that tissue can be used safely and effectively to restore future fertility (13, 16, 21, 38–41).

Table 1.

Literature reporting progress in stem cell technology development

Stem Cell Technologies	Experimental Endpointsa
	Histology/Immunological staining					Transplant			Functional readout
	(q)RT- PCR	Histology	ICC	IHC	Flow/ FACS/ MACS	Xeno	Homologous	Autologous	Spermb	Fertilization	Progeny
SSC Transplant
Rodents		(30, 31, 48–51, 57, 58, 118, 119)		(71, 120)		(71, 118–120)	(30, 31, 48–51, 57, 58, 70, 121)		(30, 31, 48, 50, 51, 57, 58, 71, 118–121)	(31, 48, 50, 51, 57, 58, 71, 120, 121)	(31, 48, 50, 51, 57, 58, 71, 120, 121)
Large Animalc		(54–56)		(56, 64, 72)		(64, 72)	(52–56)	(56)	(52–55)	(52, 55)	(52, 55)
Nonhuman Primate		(59, 60)		(122–125)		(122–125)	(29)	(29, 59–61)	(29, 59)	(29)
Human				(21, 38, 78, 89, 109, 115, 117)		(21, 38, 78, 89, 109, 115, 117)		(67)
SSC Culture
Rodents		(57, 58)	(70, 83)	(71)	(58, 70, 71, 121)	(71)	(57, 58, 70, 71, 121)		(57, 58, 71, 83, 121)	(57, 58, 71, 121)	(57, 58, 71, 121)
Large Animal	(73)		(72–75)	(72)		(72)
Nonhuman Primate	(76)		(76)
Human	(21, 38, 79, 81)		(77, 79, 81, 82, 126)	(21, 38, 78, 80)	(77, 80–82, 126)	(21, 38, 79)
Testicular grafting
Rodents		(92, 93, 95)				(95)	(92, 93, 95)		(92, 93)	(92, 93)	(93)
Large Animal		(92)				(92)			(92)	(92)
Nonhuman Primate		(94–98)		(97, 98)		(94–96)		(97, 98)	(94, 98)	(94)
Human		(99, 101–104)		(100, 101, 103, 104)		(99–104)
Testicular Organ Culture
Rodents		(27)					(27)		(27)	(27)	(27)
Large Animal
Nonhuman Primate
Human
Pluripotent-derived male germ cells
Rodents			(28)		(28)		(28)		(28)	(28)	(28)
Large Animal
Nonhuman Primate	(32, 33)		(32, 33)
Human	(22–24, 26)		(22–26)		(22, 23, 25, 26)

^aAbbreviations: RT-PCR, real time polymerase chain reaction; ICC, immunocytochemistry; IHC, immunohistochemistry; Flow, flow cytometry; FACS, fluorescence-activated cell sorting; MACS, magnetic cell sorting; Xeno, xenotransplantation

^bSperm and spermatogenesis

^cFarm animals and companion animals

Spermatogonial stem cells and spermatogenesis

Spermatogonial stem cells (SSCs) are the adult tissue stem cells in the testes that balance self-renewing and differentiating divisions to maintain continuous sperm production throughout the postpubertal life of men. SSCs are rare cells located on the basement membrane of seminiferous tubules (Figure 2A and B). These stem cells give rise to undifferentiated progenitor spermatogonia that undergo several transit amplifying mitotic divisions, followed by two meiotic divisions and morphological differentiation to produce sperm. Spermatogenesis is an extraordinarily productive system that produces approximately 40 million sperm per gram of tissue per day in mice (42) and 5.5 million sperm per gram of tissue per day in men (43). The difference in sperm production in mice and men can be explained in part by differences in the number of transit amplifying mitotic divisions that precede meiosis (44). In humans, the spermatogonial stem cell pool is comprised of A_dark and A_pale spermatogonia. A_dark are relatively quiescent, while A_pale are mitotically active and undergo 1–2 transit amplifying divisions before giving rise to differentiating type B spermatogonia and then primary spermatocytes, which enter meiosis and migrate off the basement membrane of the seminiferous tubule (Figure 2B and C). Due to its highly proliferative nature, the spermatogenic system is an unintended target of chemo- and radiotherapies. The relative effects of various chemo- and radio-therapy regimens on male fertility are detailed in several previous reports (9, 11, 19, 45, 46). The extent that the fertility of a patient is affected, depends on the dose, type and frequency of treatment (1). There has been significant progress in minimizing the unwanted side-effects of the cancer treatment by adjusting and modifying the therapeutic regimens without compromising the efficiency of oncological treatments (47). Nonetheless, some cancer treatments, like whole body radiation, radiation to the gonads and alkylating chemotherapy, can be particularly gonadotoxic and result in prolonged or permanent azoospermia. In the following sections, we will review experimental stem cell-based approaches that may have application for preserving and/or restoring male fertility.

Figure 2.

Human spermatogonial stem cells and spermatogenesis. (A) Testes are comprised of seminiferous tubules that start and end at the rete testis. (B) Cut-out of the basement membrane of the seminiferous epithelium. (B and C) The basement membrane of the seminiferous epithelium contains undifferentiated (A_dark and A_pale) spermatogonia and differentiating Type B spermatogonia. Type B spermatogonia give rise to primary spermatocytes that enter meiosis and migrate off the basement membrane. Subsequent meiotic divisions and morphogenesis give rise to secondary spermatocytes, spermatids and the terminally differentiated spermatozoa that are released into the lumen of the seminiferous tubules

Spermatogonial stem cell transplantation

Brinster and colleagues pioneered the technique for spermatogonial stem cell transplantation in mice in 1994, demonstrating that donor SSCs could engraft the seminiferous tubules of chemotherapy-treated recipient mice and regenerate spermatogenesis and fertility leading to the production of viable progeny through normal breeding (30, 31). The SSC transplantation technique has become the experimental gold standard for quantifying stem cell activity and may have application for treating male infertility. Homologous species SSC transplantation has now been reported in mice, rats, pigs, goats, bulls, sheep, dogs and monkeys, including the production of donor-derived progeny in mice, rats, goats and sheep (29, 48–61). SSCs from donors of all ages, newborn to adult, can regenerate spermatogenesis (49, 62) and SSCs can be cryopreserved and retain spermatogenic function upon thawing and transplantation (29, 63, 64). We recently demonstrated that SSCs from the testes of prepubertal and adult rhesus macaques could be frozen, thawed and transplanted to regenerate spermatogenesis and produce fertilization competent sperm (29, 59). Progress in these previous studies suggest that it should be feasible for prepubertal boys or adult men to cryopreserve testicular tissue containing SSCs prior to treatment and have these cells reintroduced into their testes at a later date to regenerate spermatogenesis.

Radford and colleagues initially introduced the autologous SSC transplantation technique to the human clinic in 1999 (65). In Manchester, the United Kingdom, testicular tissue from 12 male non-Hodgkin’s lymphoma patients was cryopreserved as a cell suspension prior to the initiation of chemotherapy. At later dates, seven of the patients had the cells injected back into their testes (66, 67). To our knowledge, there have been no follow up reports on the fertility status of those patients so the outcome of the experiment is unknown. Even if the men in that study fathered children, it would be difficult to demonstrate unequivocally (in the absence of a unique genetic marker) that those offspring resulted from sperm produced by transplanted stem cells rather than from surviving endogenous stem cells. There have been no subsequent reports of SSC transplantation in humans since the 1999 study. Nonetheless, this bold, pioneering study demonstrated that patients are willing to pursue experimental stem cell approaches to achieve fertility (65, 67). To date, published reports indicate that testicular tissue or cells have been cryopreserved for more than 150 prepubertal and adult male patients worldwide and this is likely an underestimate of actual cases (13, 14, 21, 38–41, 65, 67).

For SSC transplantation in rodents, the testes are typically accessed via a mid-ventral abdominal incision. Testicular cells (including SSCs) are injected using a pulled glass capillary pipet inserted via the efferent ducts into the rete testis space, which can be visualized on the surface of the testis and is contiguous with all seminiferous tubules (68). Testis anatomy in larger animals, including nonhuman primates and humans is different than rodents, with the rete testis being centrally located in the testes. Schlatt and colleagues (69) demonstrated that ultrasound can be used to visualize the rete testis and guide an injection needle into the rete testis space. Ultrasound-guided rete testis injection has now been employed for SSC transplantation in several large animals species, including nonhuman primates (29, 52–56, 60, 61). For this approach, an injection needle is simply inserted under ultrasound guidance through the scrotal skin and testicular parenchyma into the rete testis space (Figure 3 depicts an example from nonhuman primates)(29).

Figure 3.

Ultrasound-guided rete testis injections. For SSC transplantation into larger animals, including nonhuman primates, (A) ultrasound is used to visualize the rete testis (echo-dense structure). (B and C) The injection needle is inserted under ultrasound guidance through the scrotal skin into the rete testis space, which is continuous with the seminiferous tubules. (C) Positive pressure is applied to the needle so the cells are slowly injected into the rete testis space and seminiferous tubules.

Clinical translation of the SSC transplantation technique appears eminent considering successes in several large animal models (Table 1) and that many patients have already cryopreserved testicular tissue or cells (detailed above). However, several questions and/or challenges remain. First, the small amount of tissue that can be obtained from testicular biopsies (especially from prepubertal boys) may contain a relatively small number of stem cells. Second, in the cancer survivor paradigm, it is essential to eliminate the risk of reintroducing malignant cells. The sections below describe approaches that may circumvent these two challenges. A third challenge for translating new stem cell technologies to the clinic is the limited availability of reagents and experimental tools for studying humans SSCs.

Spermatogonial stem cell culture

In rodents, SSCs can be greatly expanded in culture and maintain competence to produce spermatogenesis and restore fertility upon transplantation (57, 58, 70, 71). Several groups have reported extending SSC culture to large animal species (72–76). This approach could theoretically be employed to amplify the small number of stem cells that can be obtained from a biopsy and also to demonstrate the absence of malignant contamination. A number of laboratories have reported culturing human SSCs (21, 38, 77–82), including three from the testes of prepubertal patients (38, 78, 80). These human tissue studies are promising, but challenged by the inability to evaluate the full spermatogenic potential of cultured cells by homologous species transplantation into human testes. To date, each laboratory has used a different approach to culture human SSCs and different methods to assess outcomes. These published human SSC culture results need to be replicated in other laboratories and evaluated using robust markers of human spermatogonia and by xenotransplantation to nude mice (see section below on experimental methods to track human germline stem cells).

In a recent study, Elhija and colleagues reported that testicular cells from 7-day old immature mice could be expanded in a three-dimensional soft agar culture system and differentiated to produce postmeiotic germ cells, including morphologically normal sperm (83). Additional studies are needed to confirm that the resulting sperm are functionally competent to fertilize mouse oocytes. If a similar approach is successful in humans, this would eliminate the need to put cells back into the patient and thus the risk of reintroducing malignant cells into a cancer survivor.

Cell sorting strategies to isolate SSCs and remove malignant contamination

There are legitimate concerns about malignant contamination of preserved testicular tissue, especially from leukemia patients (84, 85). Several groups have demonstrated that it is feasible to isolate germ cells and remove malignant contamination from heterogeneous testicular cell suspensions using a fluorescence-activated cell sorter (FACS) and combinations of germ cell and/or cancer cell markers (86– 90). Despite these encouraging results, caution is warranted because even low levels of contamination can lead to cancer and current assays may lack the sensitivity to detect very rare malignant cells (85, 91). Therefore, alternative methods for decontamination and screening as well as methods that do not require transplantation should be considered in some cases.

Testicular tissue grafting

In contrast to SSC transplantation, which involves disaggregation of SSCs from their cognate niches, testicular tissue grafting and testicular tissue organ culture (next section) maintain the integrity of the stem cell/niche unit. Dobrinski and Schlatt demonstrated that testicular tissues obtained from newborn mice, pigs and goats could produce complete spermatogenesis when grafted under the skin of nude mice (92) and later reported the production of offspring from sperm obtained after ectopic (under the skin) grafting of mouse testicular tissue (93). Testis tissue from prepubertal rhesus macaques also produced complete spermatogenesis with fertilization competent sperm after ectopic xenografting into nude mice (94). Survival and spermatogenesis from adult testicular tissue grafts have been less successful than immature grafts (95).

Cryopreservation is an essential component of the fertility preservation paradigm. Jahnukainen and coworkers evaluated several cryopreservation strategies with immature monkey tissues and found that freezing in 1.4M DMSO provided good graft survival and spermatogenic induction up to the spermatocyte stage (96). However, haploid germ cells were not produced in any grafts from that study, fresh or frozen. Wistuba and colleagues performed autologous testicular grafting in two studies in marmoset monkeys (97, 98) and reported that complete spermatogenesis was obtained in orthotopic (in the scrotum), but not ectopic grafts. Frozen and thawed tissues were also grafted in that study, but these were only transplanted ectopically and did not produce spermatogenesis (98). Therefore, the question of whether frozen and thawed grafts can produce fertilization competent haploid germ cells still needs to be addressed.

Several groups have reported xenografting of human testicular tissue to nude mice and so far none have observed complete spermatogenesis or haploid gametes (99–104). In general, testicular tissues xenografted from prepubertal or adolescent boys survived long-term (4–12 months)(100, 101, 103, 104) with a calculated 3.7% spermatogonial recovery rate after six months in one study (104) and three studies reporting spermatocytes as the most advanced stage of germ cell development between 6 and 12 months after transplantation (101, 103, 104). Some of those tissues were already post-pubertal and contained meiotic or post-meiotic cells at the time of transplant, so it is difficult to exclude the possibility that spermatocytes or occasional spermatids that were observed had persisted from the time of transplant (101, 104). Importantly, the study by Sato and colleagues (103) observed primary spermatocytes one year after xenografting of testicular tissue from a 3 month old boy, which clearly did not contain spermatocytes prior to grafting. In contrast to prepubertal/adolescent tissues, xenografted adult testicular tissues regressed over time (99, 102). Although the human to mouse xenografting results are somewhat discouraging, the results of the monkey studies cited above suggest that autologous transplantation may be an option if suitable cryopreservation conditions are developed. Similar to SSC transplantation, autologous grafting will be problematic in cases where malignant contamination of the testicular tissue is suspected. Xenografting of human testicular tissue into animals could circumvent this problem, but is associated with additional concerns about xenobiotics and has been unsuccessful to date.

Testicular tissue organ culture

Sato and colleagues (27, 105) demonstrated that haploid male germ cells from mice could be generated in testicular tissue organ culture, using a gas-liquid interface method that was originally devised to keep differentiated organs alive in vitro (106). Testis tissue was obtained from 2.5 to 3.5 day old mice that contained only undifferentiated germ cells, similar to prepubertal patients. Testicular tissue was minced into fragments (1–3 mm diameter) that were placed on an agarose gel that was half soaked in medium, such that the tissue was exposed to air and absorbed nutrients through the agarose. Haploid round spermatids or sperm were retrieved after 23 or 42 days in culture, respectively, and used to successfully fertilize mouse oocytes in vitro by round spermatid injection (ROSI) and ICSI. The resulting embryos were transferred to recipient females and generated live offspring with normal development to adulthood and normal fertility (27). The authors also maintained frozen and thawed testicular tissue pieces in organ culture and were able to generate sperm but the fertilization potential of that sperm was not tested. If these results in mice can be translated to humans, testicular organ culture would circumvent the need to put tissues or cells back into the patient and may be a safe option for patients with malignancies that contaminate the testes.

Germ cells derived from pluripotent stem cells

For males who did not preserve sperm or SSCs prior to gonadotoxic treatment, generation of transplantable germ cells (Primordial germ cells or SSCs) or haploid gametes from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been explored. The iPSC technology is especially exciting because it would theoretically allow patients with no germ cells in their testes to father genetically related children. The derivation of germ cells from pluripotent cells will be covered in more detail in other articles in this issue. Here we will summarize briefly that several groups have reported the generation of germ cells from nonhuman primate ESCs (32, 33) and human ESCs and/or iPSCs (22–26), including evidence of rare haploid cells in some cases. These results are exciting and revolutionary, but challenged by the inability to test the function of the resulting germ cells in the human system. Therefore, safety and feasibility will need to be established in animal models. In 2011 Hayashi et al. showed that mouse ESCs and iPSCs can be induced to an epiblast-like cell that then gives rise to primordial germ cells when BMP4 was added to a culture media (28). The resulting germ cells were transplanted into the testes of infertile mice and produced spermatogenesis with sperm that were used to fertilize oocytes using ICSI. The resulting embryos were transferred to recipient females and gave rise to viable offspring. These promising results need to be replicated in other laboratories and more research is needed before this technique can be translated to the clinic, as some of the offspring developed malignant tumors around their neck area (28). For iPSC-based technologies, it will be important to understand the effects of reprogramming, culture and differentiation on the genome and epigenome of in vitro-derived germ cells compared with endogenous germ cells (107).

Experimental methods to track and quantify human germline stem cells

Studies on human cells and/or tissues are a valuable stepping stone toward clinical translation. However, these studies are challenged by the limited experimental tools for quantifying human spermatogonia and testing their function. In animal studies, the most compelling experiments are those that demonstrate the ability of a test cell population to regenerate spermatogenesis and produce fertilization competent sperm, embryos and offspring. This is an unrealistic expectation for human studies because fertilization of human eggs and production of human embryos is not universally legal or fundable. Based on progress in animal models, human to human SSC transplantation is likely to be feasible in the clinical setting, but this is not an option for routine testing of spermatogenic potential in the laboratory setting. Therefore, experimental assessment of human germline stem cell potential must rely on descriptive endpoints and xenotransplantation for functional testing.

Here we propose that reliable markers of human spermatogonia are those with expression limited to germ cells located on the basement membrane of human seminiferous tubules. Proteins that meet these criteria, based on personal experience and review of the literature include PLZF, GFRα1, GPR125, SALL4, LIN28, UCHL1, UTF1, FGFR3, EXOSC10, DSG2, CBL, SSEA4, CD9, OCT2 and SSX (77, 78, 108–116). Examples of PLZF, SALL4 and PGP9.5 expression in the seminiferous tubules of adult human testes are shown in Figure 4A–C (Valli and Orwig, unpublished data).

Figure 4.

Experimental techniques to assay human spermatogonia. (A,B and C) Expression of spermatogonia markers PLZF (A), SALL4 (B) and PGP9.5 (C) is limited to germ cells located on the basement membrane of human seminiferous tubules. Thus, they are reliable markers to screen test cell populations for human spermatogonia. DAPI (blue) stains all cell nuclei. Scale bar = 50µm. (D) Human to nude mouse xenotransplantation assay. Human testicular tissue is made into a cell suspension and then transplanted into the testis of busulfan-treated infertile nude mice. Two months following the transplantation, the testes are recovered, the tunica is removed and the seminiferous tubules are gently dispersed to make a whole mount. The tubules are then stained with anti-primate antibody (122) to recognize the colonies of human spermatogonia (green).

In rodents, SSC transplantation is the gold standard that allows investigators to quantify germline stem cells by observing their biological potential to produce and maintain spermatogenesis in infertile recipient animals. At present, human to nude mouse xenotransplantation is the best functional assay to test the spermatogonial stem cell-like potential of a test cell population (21, 38, 78, 89, 109, 115, 117). This method does not recapitulate complete spermatogenesis from transplanted cells like mouse to mouse SSC transplantation, probably due to evolutionary distance between humans and mice. However, human to nude mouse xenotransplantation does assay the ability of transplanted cells to migrate to the basement membrane of seminiferous tubules, proliferate to produce characteristic colonies of spermatogonia and persist long term (78, 89, 109, 115, 117).

Conclusions

The assisted reproduction field is on the verge of a renaissance, which is fueled in part by exciting developments that merge new stem cell technologies with fertile outcomes. Thus, it is reasonable to expect in the next decade that the options to preserve and restore male fertility will expand from sperm freezing followed by IVF/ICSI to also include stem cell transplantation, tissue grafting and/or culture to produce fertilization competent gametes (Figure 1 and Table 1). As with any rapidly developing field that has potential to impact the clinic, it is essential to establish strict criteria to monitor progress and avoid sensationalism. Responsible technology development should ideally include: 1) Studies in lower mammals (e.g., rodents and other domestic species) where it is possible to examine functional readouts such as regeneration of spermatogenesis, fertilization, embryo development and generation of healthy offspring. 2) As technologies mature, preclinical studies in nonhuman primates that have anatomy and reproductive physiology similar to humans will demonstrate conservation of biological concepts and facilitate optimization of technical/surgical approaches (e.g., biopsy, cell processing, freezing and transplantation). Nonhuman primate studies are also amenable to functional assessments, although it is recognized that such studies will be limited to institutions with space, knowledge and technical expertise for research in primates and specifically primate assisted reproductive technology. 3) Direct investigations of human cells or tissues are particularly valuable on the road to clinical translation, but are challenged by limited availability of human samples, study to study variation in sample quantity and quality and the limited functional assays to assess experimental outcomes. Despite these challenges, human tissue studies to optimize tissue and cell processing procedures, cryopreservation methods and cell/tissue culture conditions will be the most relevant to the clinics that are already preserving testicular tissue for patients. Although it is not popular in the current era, descriptive studies of human germ lineage development in situ are essential to guide experimental design and interpretation of results of human stem cell studies.

Clinics worldwide are preserving testicular tissue for patients who do not have sperm in anticipation that new male fertility technologies will be available in the future. Each technology described in this review has merits and limitations that are detailed in the sections above. Due to uncertainty about which methods will ultimately be translated to the clinic, it seems reasonable to preserve intact testicular tissue fragments in a way that maximizes viability after freezing and thawing (an active focus of research in many laboratories) and will be amenable to tissue or cell based approaches in the future. Variability in patient circumstances and reproductive goals dictate the parallel development of multiple technologies for preserving and restoring male fertility.