Optimal isolation, culture, and in vitro propagation of spermatogonial stem cells in Huaixiang chicken.

Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs in vitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.

An in vitro testicular organoid model for the study of testis morphogenesis, somatic cell maturation, endocrine function, and toxicological assessment of endocrine disruptors.

Male reproductive capacity has fallen considerably in recent decades; in addition, the incidence of testicular cancer has increased in many developed countries. The cause of this phenomenon is unknown, but environmental toxicants are considered a major contributing factor. To study potential reproductive toxicants, robust in vitro testis models are needed. We have recently established a porcine testis organoid system with a high resemblance to the architectures of innate testis tissue. Here, we further investigated the testis morphogenesis, cell maturation, and endocrine function of the testis organoids. We also challenged this system with abiraterone, a steroidogenic inhibitor, to validate its suitability as an in vitro platform for endocrine toxicology tests. Our results showed that the testis cells in the organoids reorganize into testis cordal structures, and the cordal relative areas increase in the organoids over time of culture. Moreover, the diameters and cell numbers per cross-section of the cordal structures increased over time. Interestingly, Sertoli cells in the organoids gradually underwent maturational changes by showing increased expression of androgen receptors, decreased expression of the anti-müllerian hormone, and formation of the blood-testis barrier. Next, we confirmed that the organoids respond to hormonal stimulation and release multiple sex hormones, including testosterone, estradiol, and progesterone. Finally, we showed that the production of testosterone and estradiol in this system can be inhibited in response to the steroidogenic inhibitor. Taken together, our organoid system provides a promising in vitro platform for male reproductive toxicology studies on testis morphogenesis, somatic cell maturation, and endocrine production.

Effects of Growth Factors on In Vitro Culture of Neonatal Piglet Testicular Tissue Fragments.

In vitro spermatogenesis (IVS) has important applications including fertility preservation of prepubertal cancer patients; however, thus far, IVS has only been achieved using mouse models. To study the effects of growth factors on the maintenance of testicular tissue integrity, germ cell numbers, and potential induction of IVS using a porcine model, we cultured small testicular fragments (~2 mg) from 1-wk-old piglets under six different media conditions (DMEM + 10%KSR alone or supplemented with GDNF, bFGF, SCF, EGF, or a combination of all) for 8 weeks. Overall, tissues supplemented with GDNF and bFGF had the greatest seminiferous tubule integrity and least number of apoptotic cells. GDNF-supplemented tissues had the greatest number of gonocytes per tubule, followed by bFGF-supplemented tissues. There was evidence of gradual Sertoli cell maturation in all groups. Moreover, histological examination and the expression of c-KIT (a marker of differentiating spermatogonia and spermatocytes) and STRA8 (a marker of the pre/meiotic stage germ cells) confirmed the induction of IVS in all groups. However, GDNF- and bFGF-supplemented tissue cultures had greater numbers of seminiferous tubules with spermatocytes compared to other groups. In conclusion, overall, GDNF and bFGF supplementation better maintained the tissue integrity and gonocyte numbers and induced IVS in cultured testicular tissues.

Intra-ovarian injection of bone marrow-derived c-Kit+ cells for ovarian rejuvenation in menopausal rats.

Introduction: Cell-based therapies with certain cell types are touted as novel and hopeful therapeutic intervention in the clinical setting. Here, we aimed to assess the regenerative potential of c-Kit+ cells in the rejuvenation of ovarian tissue and fertility rate in rat model of premature ovarian failure (POF). Methods: Rats were treated with 160 mg/kg/BW of 4-vinylcyclohexene dioxide for 15 days. Freshly enriched rat bone marrow-derived c-Kit+ (MACS) and c-Kit- cells (4×105 cells/10 µL) were transplanted into the ovaries of treatment and control animals. Prior to transplantation as well as 2, 4, 6, and 8 weeks post-transplantation, randomly-selected rats were euthanized and ovarian tissues were subjected to pathophysiological examinations and real-time PCR analyses. Results: POF status was confirmed by the presence of pathological features and a decreased number of immature and mature follicles compared with the control group (P < 0.05). Histological examination revealed a substantial reduction of atretic follicles in POF rats receiving c-Kit+ cells in comparison with POF rats that did not receive these cells (P < 0.05). Compared with the control samples, angiogenesis-related genes, Angpt2 and KDR, showed increased and decreased expressions in POF ovaries, respectively (P < 0.05). c-Kit+ cells had potential to restore angiogenesis in the ovarian tissue within normal ranges. Systemic levels of FSH did not significantly change in pre- or post-transplantation time points for any group (P > 0.05). Notable reduction of collagen deposition was found in c-Kit-treated rats. Transplantation of c-Kit+ cells also restored the reduced fertility rate (P < 0.05). Conclusion: The administration of c-Kit+ cells can modulate angiogenesis and pathological changes, leading to the rejuvenation of ovarian function of a rat model of premature menopause.

Culture media and supplements affect proliferation, colony-formation, and potency of porcine male germ cells.

Gonocytes are germline stem cells in the neonatal testis with important potential applications in fertility restoration and transgenesis. Using stepwise experiments, we examined the effects of different media combined with fetal bovine serum (FBS) and/or knockout serum replacement (KSR) on the in vitro proliferation, colony-formation, ultrastructure, and expression of pluripotency markers of porcine gonocytes. Testis cells from 1-wk-old piglets were cultured for 28 days in 6 different culture media (DMEM, DMEM/F12, GMEM, α-MEM, StemPro, and RPMI), each supplemented with 5%, 10%, or 15% FBS and/or 5%, 10%, or 15% KSR. The media and FBS/KSR combination leading to the maximum number of gonocytes, and their colonies were selected for further analyses. KSR supplementation resulted in a reduced somatic cell propagation and increased gonocyte colony formation (P < 0.001). Culturing in DMEM+15%FBS led to the greatest number of gonocytes (P < 0.001), while the largest diameter and greatest number of colonies were formed in DMEM+5%FBS+10%KSR cultures (P < 0.001). Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. KSR alone did not support gonocyte propagation, likely due to a reduced somatic cell proliferation; however, the combination of FBS and KSR increased gonocyte colony formation and their size.

Using a testis regeneration model, FGF9, LIF, and SCF improve testis cord formation while RA enhances gonocyte survival.

Implantation of testis cell aggregates from various donors under the back skin of recipient mice results in de novo formation of testis tissue. We used this implantation model to study the putative in vivo effects of six different growth factors on testis cord development. Recipient mice (n = 7/group) were implanted with eight neonatal porcine testis cell aggregates that were first exposed to a designated growth factor: FGF2 at 1 µg/mL, FGF9 at 5 µg/mL, VEGF at 3.5 µg/mL, LIF at 5 µg/mL, SCF at 3.5 µg/mL, retinoic acid (RA) at 3.5 × 10-5 M, or no growth factors (control). The newly developed seminiferous cords (SC) were classified based on their morphology into regular, irregular, enlarged, or aberrant. Certain treatments enhanced implant weight (LIF), implant cross-sectional area (SCF) or the relative cross-sectional area covered by SC within implants (FGF2). RA promoted the formation of enlarged SC and FGF2 led to the highest ratio of regular SC and the lowest ratio of aberrant SC. Rete testis-like structures appeared earlier in implants treated with FGF2, FGF9, or LIF. These results show that even brief pre-implantation exposure of testis cells to these growth factors can have profound effects on morphogenesis of testis cords using this implantation model.

Brief exposure of neonatal testis cells to EGF or GDNF alters the regenerated tissue.

We have previously shown that implantation of testis cell aggregates under the back skin of immunodeficient mice results in de novo regeneration of testis tissue. We used this unique model to investigate the effects of epidermal growth factor (EGF) and glial cell-derived neurotrophic factor (GDNF) on testis cord development. Neonatal piglet testis cells were briefly (<1 h) exposed to either low (L: 0.02 µg/mL) or high (H: 2 µg/mL) doses of EGF, GDNF, or vehicle (control), before implantation in recipient mice. Randomly selected implants were removed from each mouse at 1, 2, 4, and 8 weeks post-implantation. GDNF-L implants showed increased testis cord development over time, and EGF-L implants had increased cross-sectional area. The ratio of regular cords decreased over time in EGF-H and GDNF-H implants and was replaced by a higher ratio of irregular cords in GDNF-H. EGF-L and GDNF-H implants were quickest to display rete testis-like structures. Overall, the lower dose of each growth factor was more effective than its higher dose in improving the implantation outcomes. This is the first comprehensive assessment of these key growth factors on de novo formation (regeneration) of testis tissue. Lay summary: In recent decades, testicular cancer rates have quadrupled in young men while sperm counts have dropped by half. Both conditions may be related to exposure of fetuses or infants to noxious substances causing disruption of normal testis development. To study the effects of any putative factor on testis development, we established an animal model of testis tissue regeneration. We collected newborn piglet testes after routine castration, used enzymes to completely dissociate testis cells, exposed the cells to two key growth factors (EGF or GDNF), and implanted the cells under the back skin of recipient mice, acting as live incubators. We then examined implant samples after 1, 2, 4, or 8 weeks and assessed testis regeneration. Overall, the high dose of each growth factor had adverse effects on the formation of normal testis. Therefore, this novel implantation model may also be used to study the effects of potentially harmful substances on testis development.

Culture supplementation of bFGF, GDNF, and LIF alters in vitro proliferation, colony formation, and pluripotency of neonatal porcine germ cells.

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7 days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10 ng/mL bFGF and 10 ng/mL bFGF + 100 ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on the growth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation.

Long-Term In Vitro Maintenance of Piglet Testicular Tissue: Effects of Tissue Fragment Size, Preparation Method, and Serum Source.

Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum­FBS­or knockout serum replacement­KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks.

Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties.

Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications.

Macroscopic, Histologic, and Immunomodulatory Response of Limb Wounds Following Intravenous Allogeneic Cord Blood-Derived Multipotent Mesenchymal Stromal Cell Therapy in Horses.

Limb wounds are common in horses and often develop complications. Intravenous multipotent mesenchymal stromal cell (MSC) therapy is promising but has risks associated with intravenous administration and unknown potential to improve cutaneous wound healing. The objectives were to determine the clinical safety of administering large numbers of allogeneic cord blood-derived MSCs intravenously, and if therapy causes clinically adverse reactions, accelerates wound closure, improves histologic healing, and alters mRNA expression of common wound cytokines. Wounds were created on the metacarpus of 12 horses. Treatment horses were administered 1.51-2.46 × 108 cells suspended in 50% HypoThermosol FRS, and control horses were administered 50% HypoThermosol FRS alone. Epithelialization, contraction, and wound closure rates were determined using planimetric analysis. Wounds were biopsied and evaluated for histologic healing characteristics and cytokine mRNA expression. Days until wound closure was also determined. The results indicate that 3/6 of treatment horses and 1/6 of control horses experienced minor transient reactions. Treatment did not accelerate wound closure or improve histologic healing. Treatment decreased wound size and decreased all measured cytokines except transforming growth factor-ß3. MSC intravenous therapy has the potential to decrease limb wound size; however, further work is needed to understand the clinical relevance of adverse reactions.

Testicular Changes of Honey Bee Drones, Apis mellifera (Hymenoptera: Apidae), During Sexual Maturation.

The normal developmental anatomy and histology of the reproductive tract of the honey bee drone, Apis mellifera (Linnaeus, 1758), has been well documented. The post-emergence maturation changes of the accessory glands are likewise well understood, but the normal histological changes of the testicle undergoing physiologic atrophy are not well characterized. To address this knowledge gap, herein we describe the anatomy and sequential histological stages of normal testicular atrophy of drones sampled daily from emergence to sexual maturity in the spring (June) and early summer (July). Testicular histological changes during maturation are characterized by the following stages: I) conclusion of spermiogenesis; II) evacuation of spermatodesms from tubular lumens; III) progressive follicular cell atrophy, and IV) complete atrophy and collapse of testicular parenchyma. Tubular changes occur in a basilar to apical direction where segments closer to the vas deferens are histologically more mature than corresponding apical segments. In addition, the rate of testicular maturation was found to change with seasonal progression. This description of physiologic testicular atrophy should be useful for future studies investigating potential pathological effects of stressors on drone testes during sexual maturation.

Generation of a Highly Biomimetic Organoid, Including Vasculature, Resembling the Native Immature Testis Tissue.

The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air-liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications.

Current progress, challenges, and future prospects of testis organoids†.

Spermatogenic failure is believed to be a major cause of male infertility. The establishment of a testis organoid model would facilitate the study of such pathological mechanisms and open the possibility of male fertility preservation. Because of the complex structures and cellular events occurring within the testis, the establishment of a compartmentalized testis organoid with a complete spermatogenic cycle remains a challenge in all species. Since the late 20th century, a great variety of scaffold-based and scaffold-free testis cell culture systems have been established to recapitulate de novo testis organogenesis and in vitro spermatogenesis. The utilization of the hydrogel scaffolds provides a 3D microenvironment for testis cell growth and development, facilitating the reconstruction of de novo testis tissue-like structures and spermatogenic differentiation. Using a combination of different strategies, including the use of various scaffolding biomaterials, the incorporation of the living cells with high self-assembling capacity, and the integration of the advanced fabrication techniques, a scaffold-based testis organoid with a compartmentalized structure that supports in vitro spermatogenesis may be achieved. This article briefly reviews the current progress in the development of scaffold-based testis organoids while focusing on the scaffolding biomaterials (hydrogels), cell sources, and scaffolding approaches. Key challenges in current organoid studies are also discussed along with recommendations for future research.

In vitro production of haploid germ cells from murine spermatogonial stem cells using a two-dimensional cell culture system.

The in vitro propagation and differentiation of spermatogonial stem cells (SSCs) has many potential applications within reproductive science and medicine. We established a two-dimensional (2D) cell culture system to proliferate and differentiate prepubertal mouse SSCs as a model capable of maximizing on a small number of donor SSCs. We also investigated the effects of retinol on in vitro SSC differentiation. Testis cells were cultured for 10 days in a serum-free medium. This produced SSC colonies which were then dissociated and sub-cultured for an additional 20 days in a differentiation medium. Before inducing differentiation, colonies expressed genes specific for undifferentiated spermatogonia (Ngn3, Plzf). After 10 days in the differentiation medium, Stra8 expression was upregulated. After 20 days, Acr expression was upregulated, indicating the completion of meiosis. Immunofluorescence, RT-PCR and flow cytometry confirmed the presence of haploid male germ cells (4.4% of all cells). When retinol was added to the differentiation medium the proportion of haploid germ cells increased (8.1% of cells). We concluded that, under serum-free culture conditions, prepubertal SSCs will generate colonies that can differentiate into haploid germ cells in a 2D culture system. These cells demonstrate a relatively high efficiency of haploid-cell production, which can be further improved with retinol.

Long-Term Monitoring of Donor Xenogeneic Testis Tissue Grafts and Cell Implants in Recipient Mice Using Ultrasound Biomicroscopy.

Testis tissue xenografting and testis cell aggregate implantation from various donor species into recipient mice are novel models for the study and manipulation of testis formation and function in target species. Thus far, the analysis of such studies has been limited to surgical or post-mortem retrieval of samples. Here we used ultrasound biomicroscopy (UBM) to monitor the development of neonatal porcine testis grafts and implants in host mice for 24 wk, and to correlate UBM and (immuno)histologic changes. This led to long-term visualization of gradual changes in volume, dimension and structure of grafts and implants; detection of a 4 wk developmental gap between grafts and implants; and revelation of differences in implant development depending on the craniocaudal site of implantation on the back of host mice. Our data support the reliability and precision of UBM for longitudinal study of transplants, which eliminates the need for frequent surgical sampling.

Homing and Engraftment of Intravenously Administered Equine Cord Blood-Derived Multipotent Mesenchymal Stromal Cells to Surgically Created Cutaneous Wound in Horses: A Pilot Project.

Limb wounds on horses are often slow to heal and are prone to developing exuberant granulation tissue (EGT) and close primarily through epithelialization, which results in a cosmetically inferior and non-durable repair. In contrast, wounds on the body heal rapidly and primarily through contraction and rarely develop EGT. Intravenous (IV) multipotent mesenchymal stromal cells (MSCs) are promising. They home and engraft to cutaneous wounds and promote healing in laboratory animals, but this has not been demonstrated in horses. Furthermore, the clinical safety of administering >1.00 × 108 allogeneic MSCs IV to a horse has not been determined. A proof-of-principle pilot project was performed with two horses that were administered 1.02 × 108 fluorescently labeled allogeneic cord blood-derived MSCs (CB-MSCs) following wound creation on the forelimb and thorax. Wounds and contralateral non-wounded skin were sequentially biopsied on days 0, 1, 2, 7, 14, and 33 and evaluated with confocal microscopy to determine presence of homing and engraftment. Results confirmed preferential homing and engraftment to wounds with persistence of CB-MSCs at 33 days following wound creation, without clinically adverse reactions to the infusion. The absence of overt adverse reactions allows further studies to determine effects of IV CB-MSCs on equine wound healing.

Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes.

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

The study and manipulation of spermatogonial stem cells using animal models.

Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.

Spermatogonial Stem Cells for In Vitro Spermatogenesis and In Vivo Restoration of Fertility.

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genes onto the next generation. SSCs also have the potential to provide important knowledge about stem cells in general and to offer critical in vitro and in vivo applications in assisted reproductive technologies. After century-long research, proof-of-principle culture systems have been introduced to support the in vitro differentiation of SSCs from rodent models into haploid male germ cells. Despite recent progress in organotypic testicular tissue culture and two-dimensional or three-dimensional cell culture systems, to achieve complete in vitro spermatogenesis (IVS) using non-rodent species remains challenging. Successful in vitro production of human haploid male germ cells will foster hopes of preserving the fertility potential of prepubertal cancer patients who frequently face infertility due to the gonadotoxic side-effects of cancer treatment. Moreover, the development of optimal systems for IVS would allow designing experiments that are otherwise difficult or impossible to be performed directly in vivo, such as genetic manipulation of germ cells or correction of genetic disorders. This review outlines the recent progress in the use of SSCs for IVS and potential in vivo applications for the restoration of fertility.