Testicular Organoids Research Articles

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

Establishment and Characterization of Testis Organoids with Proliferation and Differentiation of Spermatogonial Stem Cells into Spermatocytes and Spermatids.

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.

Spermatogenesis in mouse testicular organoids with testis-specific architecture, improved germ cell survival and testosterone production

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.

Generation of mouse testicular organoids with highly compartmentalized tubular lumen structure and their cryopreservation

Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ± 0.695, 13.347 % ± 3.102, 47.570 % ± 0.379, and 27.406 % ± 1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a “universal” testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.

P-797 Assessment of embryo development following fertilization with de novo male gametes generated in a three-dimensional culture

Abstract Study question Can de novo male gamete from embryonic stem cell contribute to the full pre-implantation development? Summary answer Gametes generated after 29 days of differentiation in our three-dimensional (3D) culture system can support consistent fertilization and normal embryo development to the blastocyst stage. What is known already In regenerative medicine, several 3D culture systems have been proposed and are capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids from mouse embryonic stem cells. However, in order to achieve successful fertilization and satisfactory embryo development, an heterologous transplantation in a host seminiferous tubule is required to allow proper spermiogenesis to obtain functional gametes. Here we attempt to perform neogametogensis in a novel 3D niche to generate de novo gamete ready to be used for ICSI. Study design, size, duration Mouse ESCs were first cultured on a gelatin coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that a normal spermatogenesis occur in 30 days, utilization of the de novo gametes was planned for day-15, 22, 29 and 36. Participants/materials, setting, methods Mouse ESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage) and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance The evaluation of germline-specific markers through immunofluorescence revealed consistent levels of Vasa expression, a ubiquitous germline marker, with percentages at 15% on day-3, 18% on day-10, 17% on day-15, 19% on day-22, 20% on day-29, and 13% on day-36. Dazl, a specific marker for spermatogonia, exhibited a peak expression of 45% on day-10 in spherified cells. Boule, expressed by secondary spermatocytes, reached its highest level of 10% on day-15 but progressively decreased to 8% on day-22. Acrosin, a marker for spermatids, initially manifested at day-10 at 2% positivity and increased to 5% on day-15, reaching 7% on day-22, and peaked at 20% on day-29. According to spermatogenic marker expression, the neogametes follows a maturational profile similar to in vivo mouse spermatogenesis. To prove neogametes competence, we inject the neogametes into mature oocytes. The control ICSI cohort achieved 89.2% fertilization and 77.8% blastocyst rates. Neogametes generated on day-15, 22, 29 and 36, achieved fertilization rates of 35.0%, 61.1%, 81.8% and 75.0%, respectively, and yielded blastulation rates of 5.0%, 16.7%, 36.4% and 8.3%, respectively. Resulting embryos had morphokinesis comparable to control up to the 8-cell stage, but a delay was observed from compaction to blastocyst hatching. Limitations, reasons for caution In spite of the ability to fertilize normally and support blastulation, efficiency rate remained suboptimal. More importantly, the ability to generate live offspring still has to be proven. Wider implications of the findings This novel 3D differentiation model is capable of generating competent gametes and obviate the need for allo-/xeno-geneic transplantation. Once reproducibility and ability to obtain healthy offspring are confirmed, this method may represent a novel treatment option for azoospermic men Trial registration number N/A

In Vitro Generation of Haploid Germ Cells from Human XY and XXY Immature Testes in a 3D Organoid System.

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

The limitations of testicular organoids: are they truly as promising as we believe?

The limitations of testicular organoids: are they truly as promising as we believe?

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.

Prior exposure to alkylating agents negatively impacts testicular organoid formation in cells obtained from childhood cancer patients.

Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)? Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy. Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples. Organoids hold promise in providing fundamental early insights in creating such platforms. However, the generation of TOs that closely resemble the innate testis, to enable a thorough monitoring of the necessary steps for germ cell differentiation and somatic functionalities, remains a challenge. We used a Matrigel-based three-layer gradient culture system to generate human TOs and to reveal whether chemotherapy exposure affects TO formation capacity and the functionality of pre- and peri-pubertal testicular somatic cells. Testicular cells of 11 boys (aged 7.7 ± 4.1 (mean ± SD) years) were assessed for TO formation in relation to previous chemotherapy exposure and SOX9 expression in histological sections of paraffin-embedded testicular tissue samples collected on the day of biopsy and compared with testicular tissue samples obtained from 28 consecutive patients (aged 6.9 ± 3.8 (mean ± SD) years). All 39 patients were part of the fertility preservation project NORDFERTIL; an additional 10 samples (from boys aged 5.5 ± 3.5 (mean ± SD) years, without an underlying pathology) in an internal biobank collection were used as controls. We obtained 49 testicular tissue samples from boys aged 0.8-13.4 years. Fresh samples (n = 11) were dissociated into single-cell suspensions and applied to a three-layer gradient culture system for organoid formation. Histological sections of another 28 samples obtained as part of the fertility preservation project NORDFERTIL, and 10 samples from a sample collection of a pathology biobank were used to evaluate the effects of prior exposure to alkylating agents on testicular samples. Testicular organoid formation was defined based on morphological features, such as compartmentalized structures showing cord formation, and protein expression of testicular cell-specific markers for germ and somatic cells was evaluated via immunohistochemical staining. Hormone secretion was analysed by specific enzyme-linked immunosorbent assays for testosterone and anti-Müllerian hormone (AMH) production. Our results revealed that 4 out of 11 prepubertal testicular samples formed TOs that showed compartmentalized cord-like structures surrounded by interstitial-like areas and increasing levels of both testosterone as well as AMH over a 7-day culture period. We observed that SOX9 expression was correlated positively with TO formation. Moreover, exposure to alkylating agents before biopsy was inversely correlated with SOX9 expression (P = 0.006). N/A. Due to the limited amount of material available, only 11 out of the 39 pre- and peri-pubertal testicular tissue samples could be used for the organoid formation experiments. The testicular tissue samples obtained from a sample collection of the internal biobank of Department of Pathology, Karolinska University Hospital were considered normal and included in the study if no testicular pathology was reported. However, detailed information regarding previous medical treatments and/or testicular volumes of the patients included in this biobank was not available. Our observations suggest that SOX9 expression may serve as a putative indicator of TO formation, indicating a critical role of Sertoli cells in promoting organoid formation, seminiferous tubule integrity, and testicular function in pre- and peri-pubertal testicular tissue. This study was supported by grants from the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.), Finnish Cancer Society (K.J.), Finnish Foundation for Paediatric Research (K.J.), Swedish Research Council (2018-03094; 2021-02107) (J.-B.S.), and Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2020-00335; 2021-00073; 2022-00317) (J.-B.S. and K.J.). Y.C. and Y.Y. received a scholarship from the Chinese Scholarship Council. J.P.A-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. R.T.M. was supported by a UKRI Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health was supported by an MRC Centre Grant (MR/N022556/1). The authors declare no competing interests.

An in vitro three-dimensional (3D) testicular organoid culture system for efficient gonocyte maintenance and propagation using frozen/thawed neonatal bovine testicular tissues

Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of an in vitro culture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for the in vitro maintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 106 cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 μm; the mean number of organoids was 5.57 ± 1.60/105 μm2 on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression of C-KIT but reduced the expression of GFRα1 at day 28 when compared to those without hormone supplements (p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishing in vitro spermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.

Towards a "Testis in a Dish": Generation of Mouse Testicular Organoids that Recapitulate Testis Structure and Expression Profiles.

The testis is responsible for sperm production and androgen synthesis. Abnormalities in testis development and function lead to disorders of sex development and male infertility. Currently, no in vitro system exists for modelling the testis. Here, we generated testis organoids from neonatal mouse primary testicular cells using transwell inserts and show that these organoids generate tubule-like structures and cellular organization resembling that of the in vivo testis. Gene expression analysis of organoids demonstrates a profile that recapitulates that observed in in vivo testis. Embryonic testicular cells, but not adult testicular cells are also capable of forming organoids. These organoids can be maintained in culture for 8-9 weeks and shows signs of entry into meiosis. We further developed defined media compositions that promote the immature versus mature Sertoli cell and Leydig cell states, enabling organoid maturation in vitro. These testis organoids are a promising model system for basic research of testes development and function, with translational applications for elucidation and treatment of developmental sex disorders and infertility.

Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids

Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 × 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.

Establishment and Optimization of an Aggregate Culture System of Testicular Cells from Marine Medaka, Oryzias dancena

Although testicular organoids have remarkable potential as testicular models in vitro, there have been few studies about testicular organoids in teleost fish. As a first step to establish a stable culture system for fish testicular organoids, we investigated the efficient conditions for an aggregate culture of dispersed testicular cells from adult marine medaka (Oryzias dancena) by evaluating the effects of culture methods and media composition on an aggregate culture. As the results, we found that culturing dispersed testicular cells in an ultra-low attachment 96 well without Matrigel was most effectively able to induce the formation of testicular cell aggregates among the five different methods tested. Subsequently, through media testing, we confirmed that the modified ESM2 was more optimal for this aggregate culture than the media conventionally used in porcine, human, and rat testicular aggregate cultures. Furthermore, we demonstrated that three supplements in the modified ESM2 including fish serum (FS), basic fibroblast growth factor (bFGF), and embryo extracts (EE) did not influence the number and size of the testicular aggregates formed, but fetal bovine serum and other supplements including β-mercaptoethanol, non-essential amino acids, sodium pyruvate, and sodium selenite were affected significantly. Nevertheless, the removal of three supplements (FS, bFGF, and EE) during culture negatively affected scp3 and sox9a expression levels, indicating their necessity. Finally, we identified that the sperms derived from in vitro cultured testicular aggregates were able to produce offspring after fertilization with naturally matured oocytes. The results from this study will provide fundamental information to develop the techniques for fish testicular organoid culture, which will eventually contribute to the development of reproductive biotechnology for aquaculture and the conservation of endangered fish species.

FRI443 Optimizing Conditions Of Tubule-like Structures In Testicular Organoids That Will Support Spermatogenesis

Abstract Disclosure: O. Printy: None. Testicular organoids are increasingly being investigated as a potential means of fertility restoration. These organoids have been shown to self-organize to recapitulate the inner architecture of the native testis, forming tubule-like structures that resemble the seminiferous tubules. However, these tubule-like structures lack dimensionality and do not contain a large, meiotically active germ cell population. We hypothesized that if testicular organoids are cultured under appropriate microenvironmental conditions, we will observe the development of tubule-like structures with diameters that resemble what is seen in vivo, in addition to appropriate somatic cell localization and Sertoli-germ cell interactions. To test this hypothesis, we generated organoids from 5dpp male CD1 mice at three cellular seeding densities: low density (170,000 cells per well), medium density (280,000 cells per well), and high density (310,000 cells per well). The heterogenous testicular cell suspension formed compact organoids at all three seeding densities and organoid diameter increased with increasing cell number. To assess the ability of the organoids to mimic the inner architecture of a testis, we performed immunohistochemistry on 14-day-old organoids: for the major somatic cell types of the testis Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), and Sertoli cells (SOX9). Thus far, only medium density organoids appear to recapitulate the compartmentalization seen in native testes. These organoids tend to contain semi-continuous ring-like structures composed exclusively of ACTA1positive cells; these structures encapsulate a population of SOX9-positive cells, while HSD3B2-expressing cells are located on the periphery of the organoids. To assess the organoids’ ability to fuse to form a larger aggregate, organoids were transferred to 3D printed troughs. However, organoids did not merge and appeared to die. However, when organoids are cultured together in microcentrifuge tubes and alginate structures, they fused to form a larger aggregate. In the future, immunohistochemistry will be used to evaluate whether any extended tubule-like structures formed within the aggregates. Additionally, spermatogonial stem cells will be injected into the aggregates and ELISA assays will be performed to assess the spermatogenic and hormonal functionality of the organoids. Presentation: Friday, June 16, 2023

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.

Evaluation of Apoptosis-Related Genes and Hormone Secretion Profiles Using Three Dimensional Culture System of Human Testicular Organoids

Background: In reproductive biology, testicular organoids can be used to treat infertility and to study testicular development and spermatogonial stem cells (SSCs) differentiation. Generating organoid from primary cells is challenging. In this study, testicular organoids were created using human primary testicular cells and evaluated the apoptotic gene expression and hormone secretion profiles of the organoids. Materials and Methods: Primary human testicular cells were isolated using 2-step enzymatic digestion from three brain-dead donors. Immunocytochemistry and flow cytometry analyses were performed to confirm human SSCs. Isolated cells were cultured in three experimental groups: control group (2 dimensional (2D)), group 1 (organoid culture after 2D culture), and group 2 (organoid culture immediately after enzymatic digestion). Testicular organoids were cultured in DMEM/F-12 media supplemented with follicle-stimulating hormone (FSH) and fetal bovine serum (FBS) for four weeks. After 24 hours and four weeks of culture, reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the relative expression of apoptotic genes (caspase 3, 9, Bax, and Bcl-2). At 24 hours, two weeks, and four weeks after culture, enzyme-linked immunoassay (ELISA) was used to determine the testosterone and inhibin B concentrations. Light microscopy and toluidine blue staining were also used for morphological analysis. Results: RT-qPCR results revealed that pro-apoptotic (caspase 3, 9, Bax) gene expression levels were highest in group 2 after 24 h and four weeks of culture. In contrast, the expression level of Bcl-2 (anti-apoptotic) was lower in group 2 compared to other groups. The hormone secretion levels decreased in a time-dependent manner during the cultivation. According to morphological evaluations, testicular organoids are compact, spherical structures with two to three elongated cells organized along their border. Conclusion: Our findings revealed that the testicular organoid culture system maintained hormonal secretory abilities, demonstrating the function of Sertoli and Leydig cells in the absence of testis-specific environments.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

MP01-17 THALLIUM EXPOSURE TO 3D HUMAN TESTICULAR ORGANOIDS TO STUDY miRNA BIOMARKER EXPRESSION AND DOWNSTREAM mRNA REGULATION

You have accessJournal of UrologyCME1 Apr 2023MP01-17 THALLIUM EXPOSURE TO 3D HUMAN TESTICULAR ORGANOIDS TO STUDY miRNA BIOMARKER EXPRESSION AND DOWNSTREAM mRNA REGULATION Janmejay Hingu, Adam Cohen, Nima Pourhabibi Zarandi, Anthony Atala, and Hooman Sadri-Ardekani Janmejay HinguJanmejay Hingu More articles by this author , Adam CohenAdam Cohen More articles by this author , Nima Pourhabibi ZarandiNima Pourhabibi Zarandi More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Hooman Sadri-ArdekaniHooman Sadri-Ardekani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies have demonstrated that heavy metal exposure can negatively impact male infertility and testicular carcinogenesis due to increased oxidative stress. This can lead to molecular dysfunction, part of which is mediated by small non-coding RNA known as miRNA, which may serve as potential biomarkers of disease. We present a miRNA analysis following thallium exposure to three-dimensional human testicular organoids (HTOs). METHODS: 3D HTOs were created from 2D testicular cell cultures derived from three brain-dead adult patients. HTOs were exposed to either thallium or ultrapure water. The miRNA analysis was performed using the Nanostring nCounter Human v3b miRNA panel, representing 827 unique miRNA probes. Nanostring nSolver 4.0 was used to execute target normalization with the geometric mean of the top 100 miRNAs detected, followed by background subtraction of 57 counts, which was the average of exogenous negative controls in the panel plus two standard deviations. All miRNAs with average counts <5 within a sample group were excluded to ensure robust expression. Qiagen Ingenuity Pathway Analysis (IPA) miRNA target filter was used to select for experimentally observed mRNAs associated with reproductive pathology and testis cells. RESULTS: 9 miRNAs were uniquely expressed within the thallium HTOs compared to 3 in the control. Seven of the 107 miRNAs expressed in both were significantly differentially expressed (miR-574-3p, miR-374a-5p, miR-423-5p, miR-106a-5p+miR-17-5p, miR-29b-3p, and miR-26b-5p were upregulated; miR-145-5p was downregulated). However, none survived the application of the false discovery rate. IPA miRNA target filter showed that these miRNAs regulated 161 mRNAs, including BCL2, MYC, CDKN1A, CCNA2, CCND1, and E2F1. CONCLUSIONS: Certain miRNAs have previously been linked to male infertility (miR-145-5p, miR-574-3p, miR-26b-5p), asthenozoospermia (miR-423-5p), and testicular diffuse large B-cell lymphoma (miR-17-5p). Previous studies have also demonstrated that oxidative stress from thallium-containing compounds can induce apoptosis by affecting Bcl-2 (BCL2) expression and activate nucleolar stress by altering c-Myc (MYC) and p21 (CDKN1A) expression. Thallium has also been shown to modify cell cycle progression through cyclin A (CCNA2), cyclin D1 (CCND1), and E2F-1 (E2F1). Although only 4 of these miRNAs have been implicated in male infertility and testicular cancer, further studies may elucidate other miRNAs and their downstream regulation of mRNA. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e9 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Janmejay Hingu More articles by this author Adam Cohen More articles by this author Nima Pourhabibi Zarandi More articles by this author Anthony Atala More articles by this author Hooman Sadri-Ardekani More articles by this author Expand All Advertisement PDF downloadLoading ...

Klinefelter syndrome testicular organoids: Androgen production and spermatogonia differentiation in vitro

Klinefelter syndrome testicular organoids: Androgen production and spermatogonia differentiation in vitro