Emerging Technologies: In Vitro- Derived Germ Cells and Gametogenesis

Introduction

Louise Brown was born on July 25, 1978, and was the first baby born using the technology known as in vitro fertilization (IVF). Louise’s parents, John and Lesley Brown, were infertile because Lesley had blockages in her oviducts, so her eggs could not travel to her uterus or be fertilized by John’s sperm. Louise was possible because her father was able to produce sperm that could be isolated from his ejaculate, her mother was able to produce eggs that were collected from her ovaries by laparotomy, and the medical team of Patrick Steptoe (physician), Robert Edwards (researcher), and Jean Purdy (research nurse) brought those gametes together in a petri dish to achieve fertilization. An eight-cell embryo was transferred to Lesley Brown’s uterus 2.5 days later to establish a pregnancy. IVF has now produced over 8 million babies worldwide, including 1.7% of births in the United States and 4.4% of births in Europe. The 2010 Nobel Prize in Medicine was awarded for the development of IVF to Robert Edwards ( https://www.nobelprize.org/prizes/medicine/2010/press-release/ ). Forty years after the birth of Louise Brown, there are still many patients who cannot be helped by IVF or related assisted reproductive technologies because they are not able to produce mature eggs or mature sperm. Those conditions impact 1% of people in the global population, ^, which computes to 1.3 million Americans between the ages of 20 and 50. Causes of these most severe infertility diagnoses can include diseases, medical treatments, genetics, age, or other circumstances. There are new technologies in the research pipeline that may transform the future of reproductive medicine and offer fertile hope to patients with the most difficult infertility diagnoses (e.g., no eggs, no sperm).

Medical treatments for cancer, bone marrow transplantation, gender dysphoria, or other conditions can cause infertility. Adult patients have standard of care options to cryopreserve eggs, sperm, or embryos prior to treatment that can be thawed in the future and used to achieve pregnancy using established assisted reproductive technologies, such as IVF. Those options are not available to adult patients who cannot produce eggs or sperm. Examples include women who need to urgently proceed with treatment and do not have time for ovarian stimulation/egg retrieval and transgender patients who do not want to experience puberty in the gender that is necessary to mature their gonadal tissues. Those options are also not available to prepubertal children who are not yet producing mature eggs or sperm. For those patients, there are experimental options to cryopreserve the ovarian cortex with immature primordial follicles or immature testicular tissues that contain spermatogonial stem cells (SSCs, sperm precursors). Historically, these options have been provided under experimental protocol. However, the American Society of Reproductive Medicine recently advised that the experimental label can be removed from ovarian tissue freezing based on the evidence that transplantation of cryopreserved ovarian tissues has produced over 130 live births worldwide. Spermatogonial stem cell and testicular tissue transplantation are mature technologies that have been shown to regenerate spermatogenesis and produce live births in animal models. Those technologies may be ready for translation to the human clinic, but so far there are no documented live births from transplanted SSCs or testicular tissues. Moreover, these autologous transplantation approaches may not be safe for patients with leukemia, ovarian cancer, or testicular cancer due to the risk of reintroducing cancer cells back into a patient survivor. Moreover, autologous transplantation may not be appropriate for transgender patients who do not want to experience puberty in the gender that would be required to mature their gonadal tissues. This chapter describes in vitro approaches to producing mature eggs or sperm from precursor cells or immature gonadal tissues. We will approach the topic from the perspective of a cancer patient who will receive a medical treatment that can cause infertility.

Spermatogonial Stem Cell Culture

SSC transplantation is a robust technology that may be ready for translation to the human fertility clinic (e.g., for patients who cryopreserved immature testicular tissues with SSCs when they were young). However, based on our experiences in Pittsburgh and other published reports, the amount of tissue obtained by biopsy from prepubertal patients is small (30–400 mg) and may contain a limited number of SSCs. Therefore, in vitro SSC expansion may be needed prior to transplantation to achieve robust engraftment and regeneration of spermatogenesis.

In rodents, SSCs can be maintained in long-term culture with significant expansion in number, and these SSCs retain their potential to restore spermatogenesis and fertility upon transplantation. The success of SSC culture in rodents required the development of methods to isolate and enrich SSCs while eliminating testicular somatic cells that could rapidly overwhelm the cultures. In their initial report on mouse SSC culture, Kanatsu-Shinohara and colleagues plated heterogeneous testis cells from newborn mice on gelatin-coated plates. Testicular somatic cells rapidly adhered to the plates while germ cells remained floating and could be sequentially aspirated and replated onto secondary plates to gradually remove somatic cells. In contrast, Hamra and colleagues used a positive selection approach with rat pup testis cells that were plated on laminin. SSCs rapidly adhere to the laminin-coated plates and floating testicular somatic cells could be removed by aspiration. Other studies used fluorescent-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) for the cell surface marker, THY1 (CD90), to enrich SSCs and reduce contaminating somatic cells prior to culture. Development of a serum-free, defined medium facilitated the discovery of specific growth factors that were required for SSC maintenance and proliferation in culture. While Kanatsu-Shinohara used glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF), others have shown that GDNF is necessary and sufficient to support rodent SSC expansion in culture. Addition of FGF2 and/or the soluble GDNF family receptor (GFRα1) can increase the rate of SSC expansion. ^, Finally, feeder cells, such as mouse embryonic fibroblasts, are frequently needed to maintain SSCs in culture, although feeder-free culture conditions on substrates, such as laminin have been described. Spermatogonial stem cell transplantation provided a definitive bioassay to confirm the presence and number of functional SSCs in culture.

A number of laboratories around the world have reported culturing SSCs of nonhuman primates and humans. Langenstroth and colleagues reported on the isolation, enrichment, and short-term 11-day culture of adult marmoset SSCs. Adult marmoset testis cells were placed in regular tissue culture plates with minimal essential media alpha (MEMα) supplemented with 10% fetal bovine serum (FBS) at 35°C. No feeder cells or other tissue culture substrates (e.g., laminin) were used and no growth factors were added. Similar to the experience with mouse SSC cultures, they found that when heterogeneous testis cell suspensions were placed in culture, the somatic cells rapidly overwhelmed the culture. Therefore, they established a separation culture system in which floating supernatant cells were aspirated after 24 hours and placed in a secondary culture dish. Supernatant cells and attached cells were then cultured separately for 11 days. At that time, supernatant cells were collected from the supernatant cultures and attached cells were collected from the attached cultures and tested for colonization potential by primate-to-nude-mouse xenotransplantation. Monkey and human SSCs do not regenerate complete spermatogenesis after xenotransplantation to recipient mouse testes, but they do recapitulate several functions that are unique to spermatogonial stem cells. They migrate and engraft the basement membrane of recipient mouse seminiferous tubules, proliferate to produce chains and clusters of cells with a typical spermatogonial appearance (high nuclear to cytoplasmic ratio and frequently connected by intracytoplasmic bridges), and survive long-term. ^, Using primate-to-nude-mouse xenotransplantation, Langenstroth and colleagues found that the 11-day supernatant culture retained about 60% of the number of colonizing spermatogonia that were originally placed in culture on day 0, and this was eightfold higher than colonizing activity from 11-day attached cultures.

More than 20 studies on human SSC (hSSC) culture methods have been published, including three with fetal or prepubertal testicular cells. ^{, ,} Many have used differential plating on plastic, lectin, collagen, or gelatin to enrich SSCs and reduce testicular somatic cell contamination. Others have used positive or negative FACS or MACS selection alone or in combination with differential plating. Positive selection markers have included integrin subunit alpha 6 (ITGA6), CD9, G-protein coupled receptor 125 (GPR125), stage-specific embryonic antigen 4 (SSEA4), and epithelial cell adhesion molecule (EPCAM). Negative selection markers include KIT proto-oncogene (cKIT), protein tyrosine phosphatase receptor type C (PTPRC or CD45), and THY1 (reviewed in 59). It is noteworthy that THY1 is a positive selection marker for mouse spermatogonia but a negative selection marker for transplantable human spermatogonia. ^{, , ,}

Most human SSC culture studies used culture conditions similar to those originally described by Kanatsu-Shinohara and colleagues in mice, including StemPro-34 medium supplemented with various combinations of GDNF, FGF2, EGF, and LIF. Some of those studies reported significant expansion of hSSC numbers in culture, ^{, ,} while others reported a rapid decline in hSSC numbers using the same conditions. ^{, , ,} These disparate outcomes may be explained by differences in starting cell populations, culture conditions, and approaches that were employed to identify and quantify hSSCs in culture (ranging from counting total cells in culture to quantifying xenotransplantation colonizing events). Therefore, there is no consensus on the “best method” for culturing hSSCs that has been independently replicated in different laboratories and no consensus on the best methods to identify and quantify bona fide hSSCs in culture. Nonetheless, a number of research groups have reported human SSC survival and/or expansion for periods ranging from 1 week to 6 months. While the results for human SSC culture are promising, they are challenged by the inability to test the full spermatogenic potential of these cells by xenotransplantation; and autologous or homologous transplantation in humans is currently not possible. The reconstruction of the human testis microenvironment ex vivo , in part by using methods described in this chapter, will enable an assay of human spermatogenic potential.

Testicular Tissue Organ Culture

Mammalian spermatogenesis comprises three phases of germ cell differentiation: spermatogonial proliferation, meiotic recombination and reductive divisions of spermatocytes to produce haploid round spermatids, and nuclear protein replacements and dynamic transformation of cell shape of round spermatids into spermatozoa, namely spermiogenesis. The mechanism of each of these steps in detail is being elucidated mostly due to animal experiments using rodents and genetically modified mice which have contributed significantly to deciphering genetic causes of which spermatogenic impairments and their molecular mechanism. On the other hand, the mechanisms that regulate human spermatogenesis remain a profound mystery, mostly due to difficulty in obtaining human testis as experimental material and the lack of experimental tools to achieve human spermatogenesis outside the body. As a matter of fact, in vitro spermatogenesis is possible in only a few animal species. If human spermatogenesis can be regenerated in vitro , it will be used for basic research for deciphering its molecular and cellular mechanisms, as well as various practical applications. It could serve as a platform for toxicologic testing for environmental chemicals and pharmaceuticals. Furthermore, cryopreservation of testis tissue fragments of pediatric cancer patients becomes a realistic option for preserving their future fertility by producing sperm from the tissue. ^,

Spermatogenesis is a complex cell differentiation process, as stated above, and different microenvironments are needed to support each phase. These microenvironments are arranged sequentially along the length of seminiferous tubules. The seminiferous tubule is made of Sertoli cells and peritubular myoid cells. Spermatogenic germ cells reside in the seminiferous tubule and their differentiation occurs in distinct microenvironments within the tubules. Spermatogonia are located on the basement membrane of seminiferous tubules outside the blood-testis barrier. Spermatocytes migrate off the basement membrane and through the blood-testis barrier to the adluminal space where they progress through meiosis to produce haploid spermatids. Spermiogenesis occurs adjacent to the lumen and mature sperm are deposited into the lumen. It is difficult to recapitulate and coordinate those microenvironments in a cell culture system. This has been shown repeatedly in the history of research on in vitro spermatogenesis since around the 1980s. On the other hand, studies performed in earlier decades employed mostly organ culture approaches where spermatogenic cells are maintained in their cognate microenvironment of the seminiferous tubules. An original report on in vitro spermatogenesis dates back to 1920. Later in 1937, Martinovitch reported the first successful in vitro spermatogenesis in mice using newborn testis tissues, in which primitive spermatogonia developed into meiotic spermatocyte at the pachytene stage. In the 1960s, Anna and Emil Steinberger established an organ culture system to attempt in vitro spermatogenesis. Their research was comprehensive and sophisticated but it remained a challenge to surmount the barrier of the pachytene stage of meiosis under their culture conditions. In 2011, however, successful in vitro spermatogenesis in mice using an organ culture method was reported in which immature testicular tissues were maintained on an island of agar, half-soaked in culture medium ( Fig. 38.1A ).

The success of testicular tissue organ culture for in vitro spermatogenesis in mice was achieved by a simple modification of the culture medium. Culture medium usually uses biological supplements like serum, tissue extracts, serum albumin, and so forth. The modification was the replacement of FBS with Knockout Serum Replacement ^TM (KSR) or AlbuMAX, which resulted in significant improvement of spermatogenic progression beyond the pachytene spermatocyte stage. Both KSR and AlbuMAX are commercially available and used mostly for the replacement of serum. AlbuMAX is a purified from bovine serum using a chromatography method. KSR included AlbuMAX as a key ingredient and AlbuMAX was necessary and sufficient to support progression through meiosis to produce elongated spermatids.

The success of mouse in vitro spermatogenesis opened the possibility for applications in related areas of basic research as well as clinical settings. For example, in vitro spermatogenesis in testicular tissue organ culture was used for a mutant mouse testis which lacks spermatogenesis due to a Kit-ligand defect. Kit-ligand is expressed in Sertoli cells, and its membrane-bound isoform is necessary for spermatogenesis. The Kit-ligand deficiency can be treated by introducing a functional Kit-ligand gene into Sertoli cells or by transplanting germ cells from Kit-ligand deficient mice to the seminiferous tubules of a host mouse having functional Sertoli cells. These two scenarios are neither realistic nor practical in the case of human clinics. However, it was shown that the mutant testis tissue can be cultured with a high amount of Kit-ligand in the culture medium for the progression of spermatogenesis up to haploid cell formation, with which offspring were obtained with microinsemination. This was a vivid demonstration that certain types of spermatogenesis defects can be treated by modification of the testicular tissue organ culture method. It was also shown that the SSCs can be transplanted into the empty seminiferous tubules of recipient mice and then placed in organ culture to achieve in vitro spermatogenesis. The transplanted donor SSCs migrated to their niche on the basal lamina and initiated spermatogenesis. This result demonstrated that the cultured testis tissue works almost in the same manner as the testis in vivo , accepting donor stem cell colonization and supporting spermatogenesis.

The principle of the organ culture method is to place the tissue at the interface between the gas (air) and liquid phases ( Fig. 38.1A ). The advantage of this method is the improved oxygen supply. Assuming that cells are cubes with 10 μm sides and a volume of 1000 μm, even a tissue fragment as small as 1 mm ³ is an aggregate of about 10 ⁶ cells. The amount of oxygen required by individual cells varies greatly depending on the cell type and activity state (mitotic proliferation, quiescence, etc.) but is approximately 10 to 100 × 10 ⁻¹⁸ mol/cell/second(s), except for hepatocytes, which have particularly high oxygen consumption. Oxygen consumption in testicular tissue is not small considering germ cell proliferation and meiosis. Here, let us estimate 50 × 10 ⁻¹⁸ mol/cell/sec. Then, the amount of oxygen required by this tissue fragment is 50 pmol/sec [= 50 × 10 ⁻⁶ (pmol/sec/cell) × 10 ⁶ (cell)]. If these cells are flattened (cubes 2.5 μm thick and 20 μm on a side) and arranged in a single layer at the bottom of the culture dish, they will spread over an area of 4 cm ² [20 × 20 × 10 ⁶ μm ² = 4 × 10 ⁸ μm ² ]. Oxygen dissolves from the gas phase into the culture medium (maximum dissolved oxygen concentration; 160 × 10 ³ pmol/mL) and reaches the cells by diffusion. This diffusion flow is 64 pmol/sec/4 cm ² at maximum, as calculated by Fick’s first law ( Fig. 38.1B ). This means that the above-mentioned oxygen requirement of 50 pmol/s can be met. However, if the cells are not arranged in a single layer but in a hemispherical mound of tissue, the surface area of the sphere is 3.84 mm ² (0.0384 cm ² ); thus, the diffusion flow that reaches the aggregate is reduced by more than 100-fold. This is a rough estimate, but appears to capture the actual situation quite well. So, how can we supply sufficient oxygen to all the cells in this hemispherical cell mass? We cannot increase the oxygen concentration 100-fold, but we can reduce the thickness of the culture medium layer from 2 mm to about 0.02 mm (20 μm), which is 1/100 of the original thickness. This increases the diffusion flow of oxygen by a factor of about 100 ( Fig. 38.1B ). This is the principle of the gas-liquid interface method, which also worked in testis tissues for inducing spermatogenesis.

For in vitro spermatogenesis, some modifications were introduced to improve efficiency and length of time tissues could be maintained in culture. One modification was the introduction of a microfluidic system that was originally applied in semiconductor technologies and spread into biological areas by introducing a polydimethylsiloxane (PDMS) material. PDMS is biofriendly and allows for high permeability of oxygen. Mouse testis tissues placed in a microfluidic device with PDMS and continuous flow of medium past the tissue maintained spermatogenesis for over 6 months. The advantage of PDMS was shown in a simpler way to cover the cultured tissue with a flat chip of PDMS having a shallow dent for tissue-placing space. This PDMS ceiling method improved spermatogenic progression.

The success of mouse in vitro spermatogenesis using the testicular tissue organ culture method required supplementing the basal medium with AlbuMAX. However, the difference between AlbuMAX and FBS or other biological supplements was not clearly understood. It was clear though that AlbuMAX—but not FBS—contains some important ingredients for spermatogenesis. Although AlbuMAX is basically a purified albumin, it contains various substances derived from the bovine serum. These substances combined must be everything necessary for spermatogenesis, considering the fact that the culture medium that consisted only of αMEM and AlbuMAX was sufficient for the progression and maintenance of mouse spermatogenesis.

Retinoic acid is necessary for spermatogonial differentiation and initiation of meiosis. AlbuMAX contains retinoic acid, and retinoic acid was critical for in vitro spermatogenesis. ^, AlbuMAX also contains triiodothyronine (T3) and low concentrations of LH, FSH, and testosterone. When these four hormones were tested in vitro using testicular tissue from newborn mice, T3 was most critical for the induction of spermatogenesis by inducing maturation of Sertoli cells. Testosterone showed some effects on promoting spermatogenesis leading to increased production of meiotic cells, while the effects of LH and FSH were marginal.

In addition to RA and hormones, AlbuMAX contains abundant lipids, including free fatty acids, phospholipids, and lysophospholipids. Lysophospholipids, in particular, are important for testis tissue maintenance and spermatogenesis. It was also demonstrated that antioxidative agents, vitamin E, vitamin C, and glutathione were critical for protecting germ cells from oxidative stress in culture. Although these antioxidants were not detected in AlbuMAX, various natural substances have antioxidative function and may be contained in AlbuMAX. With this information, it became possible to induce mouse spermatogenesis using a chemically defined medium (CDM) without using AlbuMAX or KSR, indicating that almost every factor necessary for mouse spermatogenesis was identified.

Despite the progress in mouse in vitro gametogenesis in organ culture, its application in other species has been limited. In rats, the culture medium and method useful for mice resulted in only a very limited production of haploid cells. Very recently, with several modifications to the culture conditions, including lowered oxygen concentration and adding supplements to the medium, it became possible to culture testis tissue of rats for faithful production of round spermatids. This may indicate that spermatogenesis has been tuned delicately in each species through evolution, and culture conditions will have to be optimized for each species. Studies using cats and marmosets have been reported but did not recapitulate the spermatogenesis process. ^,

There have been several reports dealing with human in vitro spermatogenesis. Some reported that the number of spermatogonia gradually decreased over time in culture. ^, Others reported haploid cells were produced by culturing immature testis tissues obtained from prepubertal boys. There is one report that culturing fetal human testis tissue produced spermatids that were fertilization competent. As indicated above testicular tissue organ culture conditions may need to be independently developed for each species and results must be independently replicated in different laboratories. Nonetheless, with the increasing number of pediatric cancer survivors who cryopreserved testicular tissues, there is a pressing need to develop reliable methods for human in vitro spermatogenesis.