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Quarterly Medical Review - Stem cells and organoids in endocrinology
Adrenocortical organoids: A promising tool for modelling human physiology and translational research
Melina Tedesco, Andreas Schedl, Yasmine Neirijnck *
Côte d’Azur University, INSERM, CNRS, Valrose Institute of Biology (iBV), Nice, France
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Keywords:
Adrenocortical organoid Pluripotent stem cells Congenital adrenal hyperplasia Adrenocortical carcinoma Adrenal development Disease modelling
ABSTRACT
The adrenal cortex is a vital endocrine organ that controls a wide range of biological parameters including metab- olism, blood pressure and immune response, through the release of distinct steroid hormones. As adrenal diseases can be life-threatening, understanding their molecular underpinnings and developing novel therapeutic approaches are important goals. Organoids have emerged as powerful research tools to study human fundamental biological processes, model diseases and develop novel therapies. However, research on adrenocortical organoids has only been reported recently, with a shift in focus from the use of immortalized cell lines to the emerging use of stem cells. Initially, forced expression of NR5A1 was used, but the field has evolved to prioritize directed differ- entiation. To date, few protocols have been reported that allow the directed differentiation of pluripotent stem cells into adrenocortical cells.This review provides the developmental background knowledge required for devel- oping such cell systems, reports on the present state of the art and discusses how the implementation of in vitro organoid/spheroid cultures in the adrenal field can expand our basic understanding of tissue function and influ- ence preclinical research.
1. Introduction
Adrenal glands are central endocrine organs that are composed of two histologically and functionally distinct tissues: the centrally located medulla, linked with the sympathetic nervous system, produces cate- cholamines, known as “fight or flight” hormones, which increase heart rate, blood pressure and glucose metabolism. The surrounding adrenal cortex is part of the hypothalamic-pituitary-adrenal (HPA) axis and the renin-angiotensin-aldosterone system (RAAS) and is functionally subdi- vided into concentric layers (zones), each harbouring the expression of a specific set of steroidogenic enzymes (reviewed in [1,2]). The outermost zona Glomerulosa (zG), located under the adrenal capsule, produces the mineralocorticoid aldosterone involved in electrolyte balance and the regulation of blood pressure. The zona Fasciculata (zF) is responsible for the production of glucocorticoids, mainly cortisol (in humans) or corti- costerone (in rodents), which regulate glucose metabolism, stress response and inflammation. Absent in rodents, the innermost zona Retic- ularis (zR) is responsible for the production of adrenal androgens, mainly dehydroepiandrosterone (DHEA) and its sulphated derivative (DHEA-S).
The zR, and thus DHEA and DHEAS production, develops during adre- narche, a period in early childhood that coincides with the development of the first pubic and axillary hair [3,4].
Given the vital roles of the adrenal glands in the body, any adreno- cortical dysfunction may give rise to significant health issues. Adrenal gland disorders present a diverse range of manifestations, each with dis- tinctive symptoms and therapeutic options.
Primary adrenal insufficiency (PAI) is a life-threating condition defined by the inability of the adrenal cortex to produce sufficient amounts of steroid hormones [5]. Symptoms include weight loss, hypo- tension, hypoglycaemia, a failure to thrive in children, and can be fatal if left untreated. Autoimmunity is the most common cause of PAI in adults, whereas autosomal monogenic defects affecting steroidogenic enzymes account for most of the PAI diagnosed in children. This group of recessive disorders is referred to as Congenital Adrenal Hyperplasia (CAH). This pathology manifests in two distinct forms: the rare, classic (severe) form and the most common, non-classic (mild) forms, with a prevalence of 1:15,000 and 1:200-2000, respectively [6]. 95 % of CAH are caused by 21-hydroxylase deficiency, which results in impaired
Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACTH, adrenocorticotropic hormone; AGP, adrenogonadal primordium; AP, adrenal primordium; CAH, congenital adrenal hyperplasia; cAMP, cyclic adenosine monophosphate; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sul- phate; DSD, differences of sex development; DZ, definitive zone; ESC, embryonic stem cell; FZ, fetal zone; hiPSC, human induced pluripotent stem cell; HPA, hypotha- lamic-pituitary-adrenal; PAI, primary adrenal insufficiency; RAAS, renin-angiotensin-aldosterone system; SF1, steroidogenic factor 1; zF, zona Fasciculata; zG, zona Glomerulosa; zR, zona Reticularis
*Corresponding author.
E-mail address: Yasmine.Neirijnck@univ-cotedazur.fr (Y. Neirijnck).
https://doi.org/10.1016/j.lpm.2025.104300
cortisol and aldosterone synthesis with a shift toward adrenal androgen production. CAH encompasses a spectrum of clinical manifestations, from the salt-wasting form with prenatal virilization (46,XX DSD), to postnatal milder virilizing forms with the potential to adversely affect fertility.
Steroid replacement therapy is the standard treatment for PAI. However, oral medication does not precisely mimic the physiologi- cal dynamics of the circadian rhythm of hormone release, nor of the HPA axis feedback regulations, and patients experience lower qual- ity of life, reduced fertility, higher risk of metabolic diseases, cardio- vascular complications, and adrenal crisis [7,8]. The development of novel gene- and cell-replacement therapies could in principle restore endogenous adrenocortical function without the negative side effects of hormone therapies and is therefore a major research focus of sev- eral labs.
In contrast, hypersecretion of the adrenal cortex, such as in primary aldosteronism and adrenocorticotropic hormone (ACTH)-independent Cushing syndrome, leads to symptoms such as hypertension, cardiovas- cular damage, metabolic disorders, and immunosuppression. These con- ditions usually result from bilateral adrenal hyperplasia, adrenocortical adenoma (ACA) or adrenocortical carcinoma (ACC) [9,10], with women affected up to five times more frequently than men ([11-13] and reviewed in [14]). Treatments are adapted to the type of disease, the type of symptoms and to the pathogenic mechanisms, and consists of tumour resection, adrenalectomy, corticoid receptor antagonists, ste- roidogenesis inhibitors and/or glucocorticoid supplementation [15,16]. Whereas benign ACA are relatively frequent, ACC are rare (0.7-2.0 cases per million per year) and have poor prognosis, with a 5-year sur- vival rate ranging from 13 to 80 % depending on the stage of the disease at presentation [17]. Complete surgical resection is the only curative treatment but is limited to only ~50 % of the patients (localized ACC), and recurrence is observed in 60 % of operated patients. Adrenolytic therapy with the adjuvant mitotane is a common treatment following resection. However, this adjuvant therapy is controversial because of serious side-effects such as adrenal insufficiency and neurotoxicity, and of conflicting results regarding disease-recurrence [18]. Developing novel approaches to treat ACC is therefore an important goal. Over the last years, genomic studies have provided a better understanding of the genetic defects and associated pathogenic mechanisms underlying secreting-ACA and ACC [16,19-21] . This allows better management of patients and have paved the way for targeted treatments. Therefore, as the molecular events leading to ACA and ACC differ between patients and between sexes [12,13], personalized treatments are being discussed. Future development of such targeted approaches will require a deeper understanding of the molecular processes leading to adrenal cancers in human and would greatly benefit from a suitable platform to perform drug screening.
Organoids and spheroids have emerged as powerful research tools to study human tissues in vitro. Spheroids are three-dimensional (3D) cell aggregates allowing complexification of 2D cultures. While their limited cellular diversity makes them advantageous for high-throughput sys- tems, their ability to mimic organ structures and functions is limited. In contrast, organoids are composed of multiple cell types, are able to self- organise into complex 3D structures, and recapitulate some of the key morphological and functional features of the organ.
The versatility of organoid models lends itself to a multitude of applications. While originally developed for intestinal tissues [22], they are now available for many tissues including several endocrine organs (see this issue of QMR). The enormous potential for endo- crine organoids has recently been demonstrated in two landmark studies, in which researchers performed autologous transplantation of human induced pluripotent stem cell (hIPSC)-derived pancreatic islets in two type 1 and type 2 diabetic patients [23,24]. Strikingly, this approach restored endogenous insulin production, which repre- sents proof-of-principle that stem cell-derived endocrine tissue replacement could be an effective therapy.
Compared to other organ systems, research on adrenocortical orga- noids has been somewhat lagging behind and the first protocols for directed differentiation of pluripotent stem cells into steroid-producing cells have only been reported recently [25,26]. The purpose of the pres- ent review is to describe the developmental background knowledge required for developing such protocols, report on the present state of the art and discuss how the implementation of in vitro organoid/spheroid cultures in the adrenal field can expand our basic understanding of tis- sue function and influence preclinical research.
2. Adrenal development and homeostasis
2.1. Formation and zonation of the adrenal gland
The adrenal cortex and the somatic cells of the gonads have been considered to derive from a common mesodermal embryonic structure, the adrenogonadal primordium (AGP) characterized by the expression of the transcription factor NR5A1 (also named SF1 (steroidogenic factor 1) or AD4BP) [27,28]. In mice, the AGP is first visible at around embry- onic day (E) 9.0-9.5 as a group of coelomic epithelial cells at the inter- face of the lateral plate and intermediate mesoderm, which by E10.5 has split into two distinct primordia, the adrenal primordium (AP) and the gonadal primordium (GP). Recent studies in mice, cynomolgus monkey and human have revealed that adrenocortical and gonadal fate are in fact specified independently [29,30] challenging the classical view of a common NR5A1 + progenitor. Soon after specification, cells of the AP move dorso-medially and differentiate as the transient steroidogenic fetal cortex (or “fetal zone” (FZ)) which is morphologically distinct by E11.5 in mice and 5 weeks post-conception (wpc) in human. The fetal cortex produces Sonic Hedgehog (SHH) ligands allowing the recruitment of surrounding Gli1+ mesenchymal cells [31]. Concomitantly, neural crest-derived cells of ectodermal origin, invade the fetal cortex and even- tually differentiate as the catecholamines-producing chromaffin cells of the adrenal medulla [32,33]. From E14.5 in mice and 7 wpc in human [34], the definitive cortex (or “definitive zone” (DZ)) emerges between the capsule and the FZ and cell lineage tracing in mice has shown that cells of the DZ are descendant of Gli1+capsular cells [31,35]. The fetal adrenal gland is then composed of an inner FZ and outer DZ. Whereas the human FZ secretes high amounts of DHEA-S, a ste- roid crucial for placental oestrogen production [36], the role of the mouse FZ (also called the X-zone [35]) remains unclear [37]. In both species, the FZ disappears post-natally: soon after birth in human [36], and at the onset of puberty in male and during the first pregnancy in female mice [38].
Postnatal growth of the definitive cortex involves differentiation of capsular Gli1+ and subcapsular Shh+ progenitors into steroidogenic zG cells which migrate centripetally and transdifferentiate into zF cells [31,39]. Progenitor recruitment relies on double paracrine signals, with capsular Rspondin 3 (RSPO3) ligand activating Shh expression in sub- capsular cells, which signal back to capsular cells to induce SHH-media- tors, such as Gli1 [31,40].
Although morphological and molecular features of zonation are already visible at late gestation [36,41], full maturation of adrenocorti- cal zones is completed by around postnatal day 10 in mice and by the end of the first decade in human, a time point when the formation of the zR is completed [4,37]. Zonation is established by two antagonistic sig- nalling pathways. Canonical WNT/ß-catenin signalling, mediated by capsular RSPO3-dependant Wnt4 expression, promotes proliferation and zG identity acquisition [40,42] and blocks lineage conversion to zF [43]. Proper differentiation of the zG also requires FGF and WNT/PCP signalling [44,45]. Conversely, cAMP/PKA signalling inhibits the canon- ical WNT pathway and zG differentiation, and triggers zG to zF transdif- ferentiation [46,47]. As the zR does not exist in mice, it is currently unclear how this zone differentiates, although it has been proposed that hyperactivation of PKA signalling results in conversion of the innermost
zF into cells harbouring molecular and functional features of the zR [47].
2.2. Adrenocortical maintenance
To ensure the production of hormones throughout life, the adrenal cortex is constantly and rapidly replaced. Adrenocortical cell replace- ment occurs in a highly stereotypic manner and employs similar mecha- nisms as during development. Indeed, the adult adrenal cortex is entirely replaced every 3 months (in female mice) and involves progeni- tor recruitment, proliferation in the outer cortex, centripetal migration with lineage conversion, and finally apoptosis at the corticomedullary boundary. To date, four distinct populations of adult stem/progenitor cells have been identified, with different contributions to tissue renewal. Capsular Gli1+ and subcapsular Wnt4+ progenitors represent the major source of cortical replacement during physiological tissue homeostasis [48]. A Nestin+ population has also been reported to have the capacity to differentiate into steroidogenic cells under stress [49]. Finally, Wt1+ cells, located in the mesothelial lining of the adrenal gland, represent a long-living progenitor which is recruited to generate gonadal-like ste- roidogenic cells following gonadectomy [50].
Strikingly, adrenocortical renewal is highly sexually dimorphic. While Wnt4+ subcapsular progenitors are activated in both sexes, recruitment of capsular Gli1+ progenitors is specific to female mice. Indeed, testicular androgens negatively impact proliferation and progen- itor recruitment and as a result, adrenocortical turnover is 3 times higher in female [48]. Importantly, sexual dimorphism is not limited to tissue homeostasis but can also be found in several mouse models with adreno- cortical phenotypes, as well as in human diseases ([11-13] and reviewed in [14]). The link between differences in stem cell activity and the higher prevalence of adrenal pathologies in women is still unknown and thus requires further study, including the use of in vitro human mod- els.
Over the last decades, functional genetics, cell-lineage tracing and more recently single-cell sequencing approaches in mice have consider- ably advanced our understanding of the processes involved in mamma- lian adrenal gland formation, maturation, homeostasis and physiology. Nonetheless, discrepancies between mice and human poses several limi- tations. For instance, many developmental morphogenetic events (encapsulation, colonization of neural crest-derived cells, FZ involution) have different timing. Also, mice lack a functional zR making them unsuitable as a model to study the ontogeny of this cell type. Further- more, the mouse adrenal cortex produces corticosterone rather than cor- tisol and lack DHEA/DHEA-S production. These are only some of the differences that highlight the importance of creating models that allow studying development in each species.
3. Adrenocortical cell systems
Cellular systems are essential tools to study normal and pathological processes and evaluate cellular responses to drugs. For the adrenal cor- tex, relatively few cell systems have been developed. To investigate ste- roidogenesis, established ACC cell lines serve as practical and widely used models, complementing the use of primary adrenocortical cells.
3.1. Immortalized cell lines
Established ACC cell lines such as NCI-H295 (and related substrains), CU-ACC1, CU-ACC2, MUC-1, JIL-226 and TVBF-7 have been extensively used as monolayer culture in the past (reviewed in [51,52]). In the last years, 3D-culture conditions have been developed to generate ACC cell lines-derived spheroids, which better mimic the complexity of in vivo tumours and show increased metabolic activity, steroid production and drug response compared to 2D culture [53-55]. Notably, a range of sophisticated, standardized, and high-throughput techniques and devi- ces have been developed with ACC cell lines, which allows to some
extent studying ACC metastatic potential [54]. While such approaches are expected to improve and accelerate the advancement of drug discov- ery, the main limitation of the use of ACC cell lines is their tumorigenic origin and associated genetic and molecular changes, which hampers the study of certain aspect of normal adrenocortical physiology.
3.2. Primary cells derived from normal or diseased adrenal tissues
Primary cells, either isolated from patients or from model organisms, are a second tool employed for studies. Monolayer cultures of adrenocor- tical cells from healthy donors have been instrumental in understanding the regulation of human steroidogenesis (reviewed in [51]). Organo- typic culture of human fetal adrenal glands, either through spontaneous 3D-reaggregation [56] or as intact tissue fragments [57,58], has the advantage to recapitulate cell type heterogeneity and spatial organiza- tion as found in vivo. However, their limited availability, donor-depen- dent variability, and short lifespan in culture pose significant challenges to their routine use. Defining culture conditions that permit expansion and long-term culture of adrenocortical cells should therefore be a prior- ity for future research.
Patient-derived adrenocortical cells are also employed, as they reca- pitulate more faithfully the genetic, molecular, and cellular features which exist in the tumour. Patient-derived ACC organoids have been recently developed [59] and used as a preclinical experimental system for drug sensitivity test [60]. Patient-derived adrenocortical organoids thus hold significant promise for the development of more clinically rel- evant models, but ethical concerns associated with their long-term main- tenance in vitro pose a substantial challenge to their widespread adoption.
3.3. Pluripotent stem cell-derived cells and organoids
In contrast to patient-derived organoids that rely on the expansion of existing adrenocortical cells, the differentiation of pluripotent stem cells offers another possibility to obtain fully differentiated cells of the adre- nal. Pluripotent stem cells can be either isolated from the inner cell mass of blastocysts to give rise to embryonic stem cells (ESCs) or generated from adult cells using genetic or chemical factors to yield induced plu- ripotent stem cells (iPSCs) [61]. While subtle differences between these two cell types exist, their transcriptional profile and differentiation capacity are remarkably similar. Given the ease with which iPSCs can be generated, the majority of labs uses iPSCs for their studies.
Although protocols for the differentiation of pluripotent stem cells into a variety of organ systems are now well established, differentiation into steroidogenic cells such as the adrenal cortex has been difficult to achieve. This is likely due to our limited knowledge regarding the early stages of adrenal development in humans. As a result, researchers ini- tially turned their attention to forced expression of NR5A1 (SF1), a gene that is considered as a master regulator of steroidogenesis and thus ‘imprints’ the steroidogenic identity on a given cell type [62,63]. Using this approach, the differentiation of various human stem cell types (bone marrow-, umbilical cord-, skin-, urine-, endothelial-derived mes- enchymal stem cells, ESCs and iPSCs) into adrenocortical cells that are able to produce hormones has been reported [64-70]. Importantly, transplantation of such cells appears to be able to restore steroid produc- tion in adrenalectomized mice [71]. While these are promising results, forced expression of NR5A1 is likely to result in aberrant production of steroid hormones, which limits the use of such cells for biological studies and medical purposes.
To overcome these limitations, step-by-step differentiation protocols are presently being developed by several labs that aim to mimic in vitro the developmental stages found also in vivo. Indeed, adjustments of growth factors, morphogens, physical stimuli, and extracellular matrix are used as cues to mimic the developmental signals involved in tissue patterning, specification and differentiation. Using a protocol that drives cells towards mesoderm, subsequent inhibition of the nephrogenic fate
(WNT, FGF and ACTIVIN inhibitors) and factors such as SHH and inhibi- tors of NOTCH, Sakata and colleagues [25] have obtained cells that expressed high levels of NR5A1 and steroidogenic enzymes. The proto- col relies on culturing aggregates in low-attachment wells (floating cul- ture) followed by the transfer onto transwell filters to provide an air- liquid interface that enhances oxygen supply and potentially also pro- vides mechanical cues. Steroid hormones measurement revealed the production of DHEA and DHEA-S, produced by the fetal adrenal gland in humans. Production of glucocorticoids or mineralocorticoids was negli- gible, which may however be due to the immaturity of these cultures. It will be interesting to see whether long-term culture of these aggregates (beyond 43 days) will result in full maturation of steroidogenic cells of all adrenocortical zones or whether additional factors are required to further drive differentiation.
Our own lab has chosen a slightly different approach by harnessing the detailed knowledge of cell type specification during mouse adrenal development. Indeed, unbiased single cell transcriptomic analyses sug- gest an ontogenic relationship between newly specified adrenocortical cells and lateral plate mesoderm [29,72], consistent with improved hiPSC-derived gonadal differentiation with treatment with the laterali- zation factor BMP4 [73]. In addition, adrenal and gonadal lineage bifur- cation involves anteroposterior patterning, with adrenal cells specified more anteriorly, in both mice and human [29,30]. Therefore, we first differentiated mouse ESCs via the primitive streak towards the anterior portion of caudal mesoderm that expressed markers of both, lateral and intermediate mesoderm. To provide direct cellular interactions we next transferred cells into spherical plates that provide hundreds of 3D Micro- well structures. This setup allows cells to aggregate into small spheroids with a high degree of uniformity. Culturing these aggregates in the pres- ence of the cyclic adenosine monophosphate (cAMP) analogue 8-Br- cAMP led to a sharp increase of the expression of steroidogenic enzymes and the secretion of corticosterone (the main glucocorticoid found in mice) into the medium. Of note, steroidomics analysis revealed no sex hormones, thus demonstrating that differentiation was adrenal specific [26]. Molecular analyses indicated that aggregates consisted of both pro- genitor (CITED2, GLI1, GATA4) and steroidogenic (NR5A1, CYP11A1, CYP21A1) cell types. Importantly, when treated with angiotensin II or ACTH, the organoids secreted mineralocorticoids or glucocorticoids, respectively, indicating that they respond to physiological stimuli. While these results are encouraging, there remain a number of open questions that are presently addressed. How do cells behave in long term cultures, are they able to be passaged, frozen and defrosted? Secondly, how do they behave in transplants, are they able to survive long-term engraft- ment and are they sufficient to complement for adrenal insufficiency in adrenalectomized mice? Finally, can we adapt the methodology to hiPSCs, which would provide a simple way to produce true adrenocorti- cal cells from human patients in vitro?
4. Potential applications of adrenal organoids/spheroids
Organoids have been shown to be highly versatile tools in other organ systems. In this last chapter we will discuss some of the potential applications that organoids may offer in the context of adrenal research (Fig. 1).
4.1. Basic research
Adrenal gland development has been primarily studied in the mouse as it offers easy access to tissues at precise time points of embryonic development and permits functional analysis using genetic approaches. While many aspects of development are conserved between mouse and human, there exist a number of striking differences in adrenal organisa- tion between species (e.g. lack of zR in mice). The adrenal organoid sys- tem will provide a tool to investigate the factors involved in human adrenal specification and differentiation in more detail and allow us to identify similarities and differences with other species. Once more
sophisticated organoids have been developed, they should also allow us to study later stages of development, including zonation, the molecular aspects of zonal conversion and basic processes of hormone production and secretion. Moreover, they will permit studying the impact of sex hormones on human adrenal tissue in more detail, an important issue given the sex bias in adrenal diseases [14,74]. Using both XX and XY cells will allow researchers to detect differences caused by sex chromo- somes, whereas treatment with sex hormones may be employed to study hormonally-induced sex differences.
4.2. Disease modelling, pharmacology and toxicology
Organoids have proven to be highly effective in modelling and studying diseases. In the context of adrenocortical organoid, it will be interesting to introduce mutations in CTNNB1, ZNRF3, TP53 or RB1 which belong to the most frequently altered genes in ACC patients [20,21]. The power of CRISPR/Cas9 mutagenesis should allow to introduce these mutations alone or in combination, and provide a valuable experimental system to evaluate how they con- tribute to ACC pathogenesis. This would allow us to compare the effect of different mutations in a given cell line, thus suppressing the effect of genetic background in different patients. On the other hand, the same mutation can be studied in organoids derived from different cell lines to address the role of genetic variation on tumori- genesis and evaluate the effect of drugs in different genetic environ- ments. ACC organoid models would therefore help the development of drug screening and personalized medicine approaches.
Establishing causality of a detected mutation for rare diseases has been a long-standing problem in human genetics. Recreating genetic alterations found in human patients in hiPSCs or correcting mutations in patient-derived hiPSCs, will allow us to directly test the effect of a given mutation for adrenal diseases such as adrenal insufficiencies and create specific models that should allow the development of personalised treat- ments. As an example, congenital adrenal hyperplasia (CAH) is caused by mutations in a variety of genes of adrenal steroidogenesis [6]. While the complete loss of CYP21A2 as a cause of CAH is well established, more subtle mutations in CYP21A2 and in other genes (e.g. CYP11B1, HSD3B2, STAR, CYP17A1, POR) are less well understood. Modelling these in organoids, can provide us with a better understanding of their effects on steroid production and perhaps help us to define new thera- peutic avenues [75].
In addition to drug screening, adrenal organoids are expected to be also a useful tool for toxicity studies, in particular with regard to endo- crine disruptors, such as hormones and plastics. Indeed, the adrenal cor- tex has been highlighted as an ‘ideal target’ for such endocrine disruptors, as it has an elevated blood flow, lipophilic structure, and presence of CYP450 enzymes producing toxic metabolites and free radi- cals [76]. Organoids can also be tools to study nanomaterial-induced toxicity as it was described for other organs with already available orga- noid models [77]. Incubation of adrenal organoids in the presence of these compounds is expected to provide important insights into how they affect adrenal biology.
To take full advantage of the organoid system, it will however be important to develop highly reliable and reproducible platforms that allow a standardized readout of a variety of parameters including orga- noid growth, cellular turnover and hormone secretion. This is essential to detect subtle differences that may have negligible impact in vitro but can have devastating long-term effects in a patient.
4.3. Cell replacement therapies
As discussed above, CAH is a monogenic recessive disorder and in principle could be repaired by correcting the mutation in adrenocortical cells using gene therapy. However, given the high turnover of the adre- nal cortex [48], “repaired” cells are likely to be rapidly replaced. Cell therapies should therefore target adrenal stem/progenitor cells, to
Basic Research
Disease Modelling
· Development and maintenance
Pharmacological inhibitors
Gene Editing
IPSCs ESCs
specification
differentiation
zonal conversion
· Biology of hormone secretion
mineralocorticoids
glucocorticoids
DHEA/DHEA-S
· Sexual dimorphism
sex hormones
Adrenocortical cancer organoid
· Hormone secretion
CAH organoid
· Differentiation
· Architecture
XX
XY
· Migration
Adrenocortical
Pharmacology, Toxicology
Organoid
Cell therapy
· Drug discovery
transplantation
Donor
or
Patient
somatic cells
· Toxicology testing
· Toxicity testing
reprogramming
expansion
8
differentiation + gene repair
iPSCs
adrenocortical cells
ensure a quasi-permanent supply of ‘repaired’ adrenocortical cells throughout life. While for the adrenal cortex this is at present science fic- tion, cell replacement therapies are starting to show promising results in other fields such as diabetes [23,24] and blindness [78]. Furthermore, this approach is applicable to conditions such as adrenal insufficiency, which are characterised by reduced production of steroid hormones and may benefit patients that have undergone adrenalectomies as the result of adrenal tumours. Given their endocrine function, adrenal organoids could be implanted in devices that thwart immune rejection but allow response to endogenous stimuli (e.g. ACTH/angiotensin II) and corticoid secretion.
4.4. Limitations
As we have seen, the development of adrenocortical organoids is in full swing and research in the coming years is likely to result in protocols that drive differentiation of human iPSCs into mature adrenocortical organoids. The higher throughput, lower costs and the possibility to study human material rather than another species are clear advantages over animal models. However, a series of challenges remain to be con- sidered. Batch-to-batch differences of molecules, media and matrix can jeopardize reproducibility of organoid production, and standardization will be key for a broad adoption of this technology. The high cost of
small molecules and growth factors is another challenge to opening up to a wider research community.
5. Conclusions and perspectives
In the future, it will be imperative to enhance the heterogeneity and complexity of adrenocortical organoids and replicate the cell microenvi- ronment of the adrenal gland in vivo. A number of experiments have demonstrated that the vascularisation of organoids, derived from tissues such as the brain, heart and liver, enhances the physiological relevance of the model, as well as accelerating the rate of development and over- come size limitations [79-82]. As the adrenal gland is a highly vascular- ized organ, integration of endothelial cells is particularly relevant and may enhance steroid production [83]. Several approaches are presently being explored to improve vascularization in other organ systems [84]. Of particular interest, is the development of microfluidic platforms (organs on a chip), as they have been shown to permit endothelial con- nections with organoids and intravascular perfusion to these structures [85].
The adrenal cortex does not work in isolation but forms tight interac- tions with the adrenal medulla [86]. An attempt to model these interac- tions has recently been made by combining medullary and cortex tumour cell lines [87]. By combining adrenocortical organoids with
medullary stem cells, we can envisage building more complex organoids that can be used to further explore signalling between these intertwined tissues. In the long run, even more complex arrangements could be envisaged that would model inter-organ interactions of the HPA axis. A protocol that resulted in the generation of hypothalamic-like neurons is available [88] and hypothalamic-pituitary functional units can be gener- ated from 3D-cultured human iPSCs [89].
In summary, while adrenal organoids are still only in their infancy, their potential applications to improve our understanding of adrenal dis- eases and developing new therapeutic strategies are enormous. Given their ability to react to physiological stimuli such as ACTH and angioten- sin II, and the development of encapsulation devices for endocrine cells, we are convinced that clinical applications are just a matter of time. These are indeed exciting times for adrenal research.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
CRediT authorship contribution statement
Melina Tedesco: Visualization, Writing - original draft, Conceptual- ization. Andreas Schedl: Writing - original draft, Funding acquisition, Conceptualization. Yasmine Neirijnck: Visualization, Writing - origi- nal draft, Conceptualization, Supervision, Funding acquisition.
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