Regulation of Cell Types Within Testicular Organoids

Abstract

Organoids are 3-dimensional (3D) structures grown in vitro that emulate the cytoarchitecture and functions of true organs. Therefore, testicular organoids arise as an important model for research on male reproductive biology. These organoids can be generated from different sources of testicular cells, but most studies to date have used immature primary cells for this purpose. The complexity of the mammalian testicular cytoarchitecture and regulation poses a challenge for working with testicular organoids, because, ideally, these 3D models should mimic the organization observed in vivo. In this review, we explore the characteristics of the most important cell types present in the testicular organoid models reported to date and discuss how different factors influence the regulation of these cells inside the organoids and their outcomes. Factors such as the developmental or maturational stage of the Sertoli cells, for example, influence organoid generation and structure, which affect the use of these 3D models for research. Spermatogonial stem cells have been a focus recently, especially in regard to male fertility preservation. The regulation of the spermatogonial stem cell niche inside testicular organoids is discussed in the present review, as this research area may be positively affected by recent progress in organoid generation and tissue engineering. Therefore, the testicular organoid approach is a very promising model for male reproductive biology research, but more studies and improvements are necessary to achieve its full potential.

Organoids are 3-dimensional (3D) in vitro models of organs. They bear either complete or partial structural and functional similarities to organs in vivo and can be generated from pluripotent stem cells, adult stem cells, or tissue-specific primary cells (1). Organoids with organotypic architecture and functions have been successfully derived from organ systems such as the intestine (2), liver (3), and brain (4, 5). These biomimetic features make organoids a valuable tool to study the testicular microenvironment in vitro (6, 7).

For our search strategy, we conducted a literature search in PubMed using the keyword “testicular organoid” to select articles reporting and discussing the reorganization of testicular cell suspensions into 3D organoids. We also searched the reference lists of identified papers for further articles. Papers published before November 10, 2020, were included in this review.

The mammalian testis has a complex tissue architecture that is composed of seminiferous epithelial and interstitial compartments (8). The seminiferous tubules contain Sertoli cells and germ cells and are lined by an extracellular matrix (ECM)-rich basement membrane that is surrounded by peritubular myoid cells (9). The interstitial compartment is primarily composed of Leydig cells, the steroidogenic cells responsible for androgen production (10, 11), which is essential for spermatogenesis. Sertoli and peritubular myoid cells both produce a number of different growth factors (12-15) that are required for germ cell self-renewal and differentiation into sperm (9).

It has been estimated that 9% to 16% of men suffer from infertility worldwide (16) and spermatogenesis is highly dependent on the spatial distribution of the testicular somatic and germ cells within the germ cell niche (17, 18). An in vitro biomimetic model of the germ cell niche would allow better understanding of the pathophysiology of infertility. Although organoid research has increased over the last decade, it is only very recently that this field has entered the area of male reproduction and different groups have put forward methods for testicular organoid generation (19-27). These studies report organoids of varying testicular architectures and functions, paving the way for applications in the field of male reproduction. Testicular organoids enable the study of testicular development and tubular morphogenesis and can be used as a model for assaying the effects of different experimental factors and treatments that impair testis formation (1, 23, 25), while patient-specific organoids could potentially produce gametes and treat infertility (19, 25). Similar to other organoid systems, testicular organoids can also be used for toxicity screening in a high-throughput manner (21, 23, 28). In this review, we will discuss how key testicular cell types are regulated within different organoid models, how they respond to the overall niche microenvironment and stimuli, and how each of the testicular organoid models reported so far may be used in biomedical research.

Sertoli Cells

For the testis to achieve its main functions, hormone and sperm production, a complex multicellular and dynamic microenvironment is required. The Sertoli cells are usually regarded as the testicular “nurse cells” because of their unique plasticity and intricate morphology/physiology (29). These cells are not only responsible for initiating the signaling cascades that will culminate in male gonad differentiation during early development, but also play critical functions for testicular development and in the regulation of spermatogenesis (30, 31). Therefore, Sertoli cells appear to be the main cell type involved both in organoid formation and function.

In mammals, Sertoli cells go through different maturational states throughout life and their degree of maturity is directly related to organoid assembly (25). Sertoli cells are considered immature during their highly proliferative period, which ranges from fetal life until a few weeks after birth or until around puberty, depending on the species (32). Sertoli cells enter a nonmitotic state after their functional maturation that involves the formation of the Sertoli cell barrier, a junctional complex that creates a protective and dynamic microenvironment for meiotic germ cells and sperm development (33-36). Immature Sertoli cells have high morphogenic potential; thus, organoids generated from these cells are an important in vitro system for studying testis morphogenesis and cellular interactions (1, 19, 23, 25, 37). Besides their value for research into gonadal development, organoids can be useful and robust in vitro models to test the effects and mechanisms underlying the effects of exposure to environmental and pharmaceutical toxins, such as endocrine disruptors, that are known to cause severe gonadotoxic effects, especially when exposure occurs during fetal/early life (23, 28, 38, 39). However, immature Sertoli cells cannot sustain full spermatogenesis and longer culture times with supplementation of specific factors may be needed for Sertoli cells to achieve a more mature status if in vitro spermatogenesis and sperm production is the end goal.

On the other hand, when mature Sertoli cells are used for testicular organoid generation cellular reorganization to typical testicular cytoarchitecture has not been achieved, probably because of the lack of morphogenic capacity of these mature cells (19-21, 27, 40). Despite not having specific testicular cytoarchitecture, these organoids are still interesting models for studying some aspects of Sertoli cell biology. The production of inhibin B, cytokine secretion profile, and expression of proteins present in the Sertoli cell barrier, for example, have been investigated, indicating that the cellular responsiveness to different supplementations can be evaluated as well as cellular viability after drug screening (20, 21, 24, 25, 41). Although the physical structure and cell contacts are not the same as in vivo, the interactions among Sertoli and germ cells can still be investigated in the context of the spermatogonial stem cell (SSC) niche and its regulation, and the findings can later be further evaluated in other models that more closely resemble the in vivo niche organization (27, 28, 42). Additionally, the susceptibility of testicular cells to pathogen infections, such as the Zika virus, and the mechanisms involved in the infection and potential treatments can be studied with the aid of testicular organoids (40, 43).

However, to our knowledge, the long-term goal of achieving in vitro spermatogenesis with sperm production within testicular organoids has not been reached yet, although there are some reports of meiotic/postmeiotic germ cells being detected in human and mouse models (20-22) (Table 1). As outlined earlier, for SSCs to fully differentiate into haploid gametes, a complex microenvironment is required. This would require combining the testicular-like cytoarchitecture obtained when immature Sertoli cells are used for organoid formation with the mature Sertoli cell characteristics that enable support of germ cell differentiation, such as a fully developed and functional barrier, polarized cytoplasm, epithelial organization, cellular interactions, and the specific signaling that controls meiotic progression. In the testicular organoid models developed so far, the Sertoli cells appear to remain in a partially differentiated state, which may not be effective for fully supporting meiotic progression and sperm production. For example, although the expression of Sertoli cell barrier–related proteins such as occludin, zonula occludens-1, and claudins has been described in Sertoli cells in organoids, a functional barrier junctional complex that resembles in vivo organization and ultrastructure has not been fully achieved (19, 20, 23, 25). Furthermore, the impermeability of such a barrier, as evidence of its functionality, has been shown only by Alves-Lopes and colleagues (19), but this impermeability did not improve germ cell differentiation in their rodent model, indicating that other necessary factors are still lacking. Moreover, mature Sertoli cells regulate both retinoic acid and androgen signaling that are required for spermatocyte meiotic progression and spermiogenesis (44-46). Although it has been shown that germ cells in organoids respond to retinoic acid stimulation and that testicular organoids are able to secrete testosterone, the levels of these factors and the maturational status of Sertoli cells may not have achieved the necessary complexity to regulate these pathways and sustain germ cell meiosis (19, 23, 25, 37). One aspect that should be investigated further with regard to Sertoli cell maturational status in organoids is the use of regressed testis, commonly observed in seasonal breeders and testicular cancer patients, or after hormonal suppression. Although in these cases the testis tissue is mature, the Sertoli cells in regressed testis show some characteristics that resemble immature/dedifferentiated Sertoli cells, such as proliferation capacity, protein expression profile, a disrupted Sertoli cell barrier, and capacity to reassemble (26, 47-49). Therefore, it may be possible to manipulate the culture conditions and induce the maturation of these Sertoli cells after organoid assembly, potentially obtaining conditions that could support the progression of meiosis and germ cell development.

Table 1.

Summary of testicular organoid reports

Authors, y	Species	Age	Cell types	Organotypic morphology, yes/no	Testosterone production, yes/no	Germ cell differentiation, yes/no	Reference
Baert et al, 2017	Human	Adult and 15 y	Primary Sertoli, Leydig, peritubular myoid, and germ cells	No	Yes	No	(20)
Pendergraft et al, 2017; Skardal et al, 2020	Human	Adult	Immortalized Sertoli and Leydig cells, primary germ cells	No	Yes	Yes	(21, 50)
Alves-Lopes et al, 2017	Rat	5-8, 20, and 60 d	Primary Sertoli, peritubular myoid, and germ cells	Yes	NA	NA	(19)
Sakib et al, 2019	Pig, mouse, monkey, human	7 d (pig), 8-10 d (mouse), 2 y (monkey), 6 mo and 5 y (human)	Primary Sertoli, Leydig, peritubular myoid, and germ cells	Yes	NA	NA	(23)
Edmonds and Woodruff, 2020	Mouse	5, 12, 21 d, and adult (8-16 wk)	Primary Sertoli, Leydig, peritubular myoid, and germ cells	Yes	Yes	No	(25)
Topraggaleh et al, 2019	Mouse	3-5 d	Primary Sertoli, Leydig, and germ cells	Yes	Yes	Yes	(22)
Vermeulen et al, 2019	Pig	4-7 d	Primary Sertoli, Leydig, peritubular myoid, and germ cells	Yes	Yes	No	(37)
Mall et al, 2020	Rat, human	5-9 d	Primary rat Sertoli, peritubular myoid, and germ cells, human iPSC–derived primordial germ cell–like cells	No	NA	NA	(26)

Abbreviations: iPSC, induced pluripotent stem cell; NA, not available.

Another aspect that needs to be considered is the decision to use immortalized or primary cells for organoid formation. Most studies to date have used primary cells, obtained either from fresh or cryopreserved tissue (19, 20, 23, 25) (see Table 1). Pendergraft and colleagues (21) applied another strategy using a mix of human primary germ cells and immortalized Sertoli and Leydig cells, yet it is difficult to distinguish whether the lack of testicular cytoarchitecture observed in that model is due to the cells being obtained from adult patients or the immortalization process. Another possible strategy is to use testicular cells derived from induced pluripotent stem cells (iPSCs), but the maturational status of the Sertoli cells obtained would still need to be evaluated according to the differentiation protocol. To our knowledge, there is no report of testicular organoids generated from pluripotent stem cells to date, although this strategy has already been reported for other organs such as the liver, brain, and kidney (1, 3, 4, 7, 38, 50-53). If achievable, this new strategy could broaden the potential of testicular organoids for translational applications as well as contribute to model diseases that affect fertility (51, 54-56).

Germ Cells

The germ cell is the key cell type involved in spermatogenesis, the process by which male gametes are produced (9). At the foundation of this process lie SSCs, the most immature cell type of the testicular germ cells. The SSCs self-renew or differentiate to undergo spermatogenesis (9). As mentioned earlier, spermatogenesis is highly dependent on the spatial interaction of testicular somatic and germ cells (17, 18, 57). In the mammalian testis, SSCs are localized along the basement membrane in the basal compartment of the seminiferous epithelium and remain in close contact with Sertoli cells, in the specific microenvironment called the SSC niche. Sertoli and myoid cells produce factors such as glial cell line–derived neurotrophic factor (12, 13), fibroblast growth factor 2 (14), and Wnt ligands (15), which are required for germ cell homeostasis (9). In addition, Leydig and other interstitial cells as well as the vasculature are also involved in the regulation of the niche microenvironment (58-60).

A 3D organoid model designed to study germ cell biology needs to mimic completely or at least partially the SSC niche. Based on the work performed so far, it appears that testicular somatic cells are the primary drivers of in vitro morphogenesis and organoid formation (23, 61). Varying the proportion of germ cells within a reasonable limit does not impair organoid formation (23). Therefore, enrichment of germ cells within organoids at the onset of organoid generation can lead to increased germ cell presence after formation, which is useful for downstream applications of the model (23).

Germ cells cultured in an engineered niche such as 3D organoids behave more like in vivo testes when compared to germ cells cultured in conventional monolayer cultures. For example, porcine germ cells cultured in organoids experience reduced autophagic stress compared to 2D cultures (23, 62). Germ cell maintenance and differentiation depend on the relative cell associations in the testes (9). Organoids were capable to support germ cell survival (19, 25), proliferation (19), and differentiation (22, 37) in different species, such as rats, mice, and pigs (see Table 1). Even organoids that lack testis-specific tissue architecture have been reported to maintain germ cells (20, 21, 24, 25) and support meiosis as shown by the expression of protamine 1 and acrosin (21) (see Table 1). Although it remains to be seen whether these meiotic cells are fertilization competent, this still shows that testicular organoids can be used to study germ cell niche biology and germ cell differentiation.

Testicular organoids also represent a unique opportunity to study the effect of drugs, toxicants, and other experimental factors on germ cell maintenance and differentiation in a tissue-specific context (19, 23, 24). For example, Alves-Lopes et al (19) reported tumor necrosis factor α– and interleukin 1 α–mediated reduction of germ cell survival and impairment of rodent organoid formation similar to what has been observed in vivo in animals (19, 63). Our group showed that exposure to mono-(2-ethylhexyl) phthalate, an environmental toxicant, led to an increase in autophagosome numbers in porcine germ cells in a dose-responsive manner, illustrating the utility of the system for assessing germ cell toxicity (23). Germ cells in organoids experienced an attenuated retinoic acid response compared to 2D cultures, indicating a modulatory effect of the 3D niche microenvironment, similar to observations in vivo (23, 57). One exciting avenue of studying reproductive toxicity reported recently is a microfluidics-based multiorgan chip system in which miniature human testicular and liver organoids are connected via a microfluidic circuitry to better mimic the in vivo metabolic profile. This model showed that the addition of cyclophosphamide led to metabolic activation of the toxicant by the liver organoid, which then resulted in the loss of germ cells in the testicular organoid. This germ cell loss was not observed when testicular organoids were cultured alone (24), showing that such multiorganoid platforms may be more physiologically relevant for drug and toxicity screening.

Peritubular Myoid Cells and Basement Membrane

The peritubular myoid cells of the testis are key players of testicular morphogenesis during embryonic life, when the early peritubular myoid cells encircle the nests formed by Sertoli and germ cells and start depositing ECM, in conjunction with the Sertoli cells, forming the basement membrane of the seminiferous cords (64, 65). The peritubular myoid cells participate actively in the blood-testis barrier and establishment of the SSC niche, and also interact directly with Sertoli cells, mediating activity through paracrine signaling (13, 66-69). Therefore, the participation of peritubular myoid cells in the generation of testicular organoids and their microenvironment cannot be overlooked. However, there has been very little investigation regarding this particular cell type in the organoid context.

The presence of peritubular myoid cells in the testicular organoids is commonly evaluated through the expression of cytoskeleton markers, such as alpha-smooth muscle actin, or the expression of ECM proteins produced by these cells, such as collagen I or IV, fibronectin, and laminin (19-21, 23, 25) (see Table 1). The localization of these proteins in the organoids varies greatly, sometimes being seen as a widespread network of fibers with no specific pattern and at other times being observed close or around the seminiferous tubule-like structures, more similar to its in situ expression at the tunica propria (19, 23, 25, 37). It is important to mention that, although alpha-smooth muscle actin is a widely used marker for peritubular myoid cells, its expression is not exclusive to these cells because this marker is also commonly observed in endothelial cells of testicular blood vessels. Therefore, the presence of other cell types expressing this and other markers cannot be ruled out, especially when the organoid cytoarchitecture does not identify the cells by their localization.

The deposition of ECM proteins is indicative of peritubular myoid cell function, while the contractility potential of these cells as seen in vivo and in seminiferous tubule-like structures in vitro has yet to be evaluated in the testicular organoids (70-73). Additionally, it may be interesting to test and manipulate the proportion of this cell type in the primary cell population used to form the testicular organoids to better understand the potential contribution of the peritubular myoid cells in organoid reassembly and organization, as well as the influence of different ECM components in this reaggregation, similarly to the use of scaffolds (20, 22, 37, 74, 75).

Interstitial Cells

A variety of cells reside in the interstitial compartment of the testis. The Leydig cell is usually the most frequently observed cell type and is mainly responsible for androgen production, while blood and lymphatic vessels, macrophages, connective tissue, and other cell types are also present in relatively lower numbers (11). Although physically separated from the seminiferous tubules where spermatogenesis occurs, the Leydig cells can directly influence sperm production, mostly through paracrine testosterone signaling, which is essential for meiotic progression and germ cell survival (45, 76). Some groups have reported the production of testosterone from testicular organoids with and without gonadotropin hormone stimulation, and even a decline in testosterone production after viral infection, suggesting that testosterone production is a good parameter to evaluate Leydig cell function in organoids (20-22, 24, 25, 37, 43). Despite androgen production, very little is known regarding the contribution of Leydig cells to testicular organoids. Most studies show the presence of these cells using steroidogenesis-related markers (eg, StAR, 3βHSD, Cyp11a1, Cyp19a1, and INSL3) and observe that Leydig cell localization varies, sometimes at the periphery of the organoid, sometimes in the core, or randomly distributed (20-23, 25, 43, 50) (see Table 1).

One aspect that should be considered when studying testicular organoid generation is the possible effects of using different proportions of Leydig cells as well as cells at different stages of development. In most mammalian species, Leydig cells exist in different populations during development, which differ in morphology, regulation, and function, including their testosterone production capacity (77-79). In rodents, 2 populations—fetal and adult Leydig cells—are usually recognized, whereas in pigs, primates, and humans, it is suggested that there are 3 phases of Leydig cell development, although much less information is known (78-81). Therefore, it is important to consider the developmental and functional status of the Leydig cells used to generate testicular organoids because this may help us understand and expand the applicability of the model. For example, some reports show that fetal mouse Leydig cells are incapable of producing testosterone because these cells do not express the enzyme 17β-hydroxysteroid dehydrogenase, and therefore transport their product, androstenedione, to the Sertoli cells that will be responsible for the final conversion to testosterone at this point (77, 82). If this close relationship is not maintained in the testicular organoids, there may not be adequate testosterone synthesis for the progression of spermatogenesis. Similarly, different developmental stages of adult Leydig cells have distinct steroidogenic capacity and are able to produce different androgens (83, 84), which could interfere with the hormonal support of the organoid depending on the Leydig cell population that is present. Therefore, if the final end point of the organoids is to achieve sperm production, it may be interesting to have different populations of Leydig cells, or their progenitors, present or able to differentiate in the organoid, to keep the microenvironment and endocrine profile as close to in vivo as possible.

Among the other components of the interstitial compartment, the presence of endothelial cells and macrophages in testicular organoids has been identified in very few studies, using specific markers such as PECAM-1/CD31 and C1QA, respectively (23, 27). However, aside from Baert and colleagues (24), who hypothesize that macrophages may participate in the remodeling of the scaffold in their human organoid model, little is discussed about their roles and importance in the organoid microenvironment. The contributions to the SSC niche of both endothelial cells and macrophages has been described previously, and this may be an important factor demanding investigation in organoids (58, 59, 85) as well as the potential influence of these cells on the regulation of androgen production (11, 78, 86, 87). Another aspect that deserves more research is the possibility of vascularization through enrichment of the initial population with endothelial cells, as described in brain and kidney organoids (25, 42, 88, 89). This would allow the evaluation of the roles of endothelial cells and microvasculature in testis function and organoid regulation, especially considering that the lack of vascularization impairs the survival of cells and tissue architecture, and may be a limiting step for potential further clinical applications (38, 42).

Future Trends

Testicular function is highly dependent on the specific spatial arrangement of the germ cell niche (9). Most organoid systems developed so far primarily depend on the morphogenic self-organization capacity of testicular cells with little or no direct manipulation (19-21, 23, 25). Of these, only a few bear variable degrees of structural resemblance to testis tissue in vivo (see Table 1). One technique to exert more direct control over the tissue architecture of testicular organoids could be 3D bioprinting. Bioprinting involves depositing cells and biocompatible materials in layers to generate living tissues of predetermined structure and geometry. This is achieved either by printing complex scaffolds from biomaterials that are then populated with cells, or by printing complex structures directly with cells and biomaterials (90). Tissue structures such as immature miniature heart and corneal stroma has been generated using 3D-bioprinting techniques (91, 92). This technique may also be used to model testicular organoids with distinct interstitial and seminiferous epithelial compartments composed of specific cell populations. Baert and colleagues have employed 3D-bioprinting technology to design testicular constructs with prepubertal testicular cells and alginate (93). Although testicular morphology was not observed, germ cell differentiation up to spermatids was reported in the system. Further optimization would be required to generate constructs with testicular tissue architecture (93).

Sato et al and subsequently others have shown that testicular organ cultures, given the right media conditions, can support maturation of somatic cells, which in turn led to androgen production and the ability to support full spermatogenesis in rodents (94, 95). Although organoids can support androgen production, spermatogenesis in organoids remains inefficient (21, 22, 25, 37). This may be due to poorly optimized media conditions that hinder the maturation of somatic cells and germ cells. A recent report by Sanjo et al put forth a chemically defined medium containing tocopherol, ascorbic acid, glutathione, and lysophospholipids to enhance spermatogenic potential of organ cultures (96). This medium is an improvement of media conditions previously used for in vitro spermatogenesis in organ cultures (94, 96, 97) and may be built on to support spermatogenesis in testicular organoids.

Another strategy for in vitro spermatogenesis, particularly in higher mammals, may be to generate organoids from adult cells. However, adult testicular somatic cells have reduced morphogenic capacity and do not form organoids well (21, 25). Three-dimensional bioprinting technology could potentially add adult testicular cells to scaffolds to generate adult organoids. Additionally, an age-chimeric organoid generated by the combination of adult and immature prepubertal cells as described by Edmonds and Woodruff (25) may also be employed. This strategy could also improve germ cell differentiation and shorten culture duration that would invariably be required to mature prepubertal cells, especially in higher mammals such as pigs, primates, and humans. Similarly, the derivation of germ cells or germ cell–like cells from iPSCs remains inefficient (98, 99). A long-term coculture system of iPSC-derived primordial germ-like cells with testicular organoids as described by Mall et al (26) may support further differentiation and maturation of these iPSC-derived germ cells. Such systems will aid the study of gonadal development and may in the future lead to fertility preservation (75, 100). It is important to note that iPSC-derived germ cells do not always faithfully recapitulate the characteristics of native germ cells. It has been shown that iPSC-derived postmeiotic cells undergo incomplete imprinting erasure and reestablishment (101, 102) and thus at this point pose ethical and safety concerns for clinical applications.

Organoids have the potential to serve as a bridge between in vitro cultures and animal models (7). As such, there is great interest in commercializing organoids. There are currently around 19 companies that are commercializing different aspects of organoid technology (103). Well-established organoids such as brain, cardiac, and intestinal organoids generated from stem cells appear to be the most coveted for commercialization (2, 4, 103). Although the field of testicular organoids is still in its infancy, it does have the potential for commercialization. One obvious choice for commercialization would be culture media formulations. If a robust testicular organoid system can be generated from pluripotent stem cells, the culture system would have tremendous commercialization potential. Private companies often collaborate directly with academia to commercialize culture systems. One such example is the STEMdiff Cerebral Organoid kit from STEMCELL Technologies, which was developed by the company through collaboration with the Institute of Molecular Biotechnology (103). Another interesting area that can be commercialized would be mass-scale generation, storage, and supply of premade testicular organoids to research laboratories and pharmaceutical companies.

The mammalian germ cell niche is extremely complex and highly organized. The testis requires precise spatial associations and regulation of cell-cell signaling to complete its primary functions: androgen and sperm production. This has made 3D organoids an intriguing approach to re-create the germ cell niche environment. Although the field of testicular organoid research is still in its nascent stages, it has already shown potential in providing greater access and insights into germ cell niche biology and testicular cell communications.

Glossary

Abbreviations

ECM

extracellular matrix

iPSCs

induced pluripotent stem cells

SSC

spermatogonial stem cell

Additional Information

Disclosures: The authors have nothing to disclose.

Data Availability

Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study.