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De hypofyse, gelegen onderaan de hersenen, vervult een cruciale functie in de regulatie van het endocrien stelsel. Deze ‘meesterklier’ stuurt fundamentele processen aan, waaronder groei, voortplanting, immuunrespons, metabolisme en stressrespons. Wanneer de hypofyse onvoldoende hormonen produceert (o.a. door beschadiging), ontstaan ernstige pathologieën. De behandeling, levenslange hormoonsubstitutie, is suboptimaal omdat het fysiologisch pulsatiel secretiepatroon van de hypofyse hiermee niet nagebootst kan worden, en de toegediende hormonen belangrijke nevenwerkingen veroorzaken. Herstel van het beschadigd hypofyse-weefsel en/of haar functie is een aantrekkelijk alternatief. De recente ontdekking van hypofyseherstel na schade is een eerste stap in deze richting. Tijdens dit regeneratieproces zijn de lokale stamcellen geactiveerd. Om meer kennis te verwerven over de mechanismen van hypofyse-regeneratie en de rol van stamcellen in dit proces, wordt in het labo een nieuw hypofyse in vitro model, met name organoïden, ontworpen. Deze 3D structuren ontwikkelen zich in vitro onder WNT-activerende condities, en weerspiegelen het weefsel van herkomst. Deze thesis onderzocht methoden om organoïde-vorming meer efficiënt te maken en ze (langer) in cultuur te houden. Verder gingen we ook na of de organoïden een hypofyse-fenotype vertonen en kunnen gestimuleerd worden tot verdere hypofysecel-differentiatie. Organoïden werden ontwikkeld vanuit zowel normale als beschadigde hypofyse (waarin de stamcellen zijn geactiveerd), door gebruik te maken van een specifiek groeimedium met o.a. de WNT-activatoren WNT3A en RSPO1. Opvallend was dat de organoïden afkomstig van beschadigd hypofyse-weefsel hun groeicapaciteit na enkele weken verliezen terwijl de organoïden van onbeschadigd weefsel maanden in cultuur kunnen worden gehouden. De organoïden van beschadigd weefsel zijn eerder cysteachtig, waar de organoïden van onbeschadigd weefsel meer dens zijn. Om de vormingsefficiëntie van organoïden te verhogen, werd er gestart van hypofyse(stam)celclusters i.p.v. enkelvoudige cellen, maar er werden nauwelijks organoïden bekomen. Vervolgens werd de invloed van meerdere groeifactoren, in het bijzonder van WNT3A en RSPO1, nagegaan. RSPO1 bleek essentieel voor organoïde-expansie vanuit de beschadigde hypofyse, terwijl noch RSPO1, noch WNT3A nodig bleken voor de expansie van de ‘normale’ organoïden. Ten slotte werd met RT-qPCR en immunofluorescentie gevonden dat de organoïden een immatuur hypofyse-fenotype vertonen, en dat differentiatie tot hormonale cellen kan geïnduceerd worden. Verdere karakterisering en optimalisering van het hypofyse-organoïde-model zal uiteindelijk leiden tot een krachtig nieuw in vitro model voor het ontrafelen van biologie, pathologie en regeneratie van dit orgaan. De bevinding dat hormonale cellen kunnen gevormd worden is veelbelovend in de zoektocht naar een betere behandeling voor hypofysedeficiëntie (zoals celtherapie).

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The pituitary is the master endocrine gland governing normal body physiology. By regulating other endocrine glands, the pituitary steers various processes such as growth, stress, metabolism and sexual maturation. Patients with hypopituitarism suffer from a multitude of life-burdening symptoms due to insufficient production of one or several pituitary hormones. Managing this disorder currently comprises lifelong hormone replacement therapy, a treatment which is unsatisfactory because not mimicking the normal pulsatile hormone secretion pattern of the gland. Therefore, the quest remains open to find therapies that are more adequate and durable. Stem cell-driven regeneration of pituitary tissue and function may advance one possible path to follow. The pituitary has been shown to contain a population of stem cells which possess the ability to self-renew and differentiate into the various hormone-producing cell types of the gland. However, phenotype and role of these stem cells remain poorly characterized. This thesis aimed at developing a novel in vitro research model (i.e. organoids) to study pituitary stem cells and their biology. Organoids are 3D in vitro structures that self-form from the stem cells of a tissue and eventually reproduce multiple biological aspects of the organ of origin. In addition, organoids are long-term expandable, thus representing powerful research tools to explore tissue (stem cell) biology. First, we established organoid cultures from adult mouse pituitary. The organoids originate from the pituitary SOX2+ stem cells, are long-term expandable, display a stemness phenotype which is retained during the expansive culture and show specific hormonal differentiation capacity, although limited, after subrenal transplantation in immunodeficient mice. Second, we applied the developed organoid protocol to transgenically injured pituitary known to harbor an activated stem cell population. More numerous organoids develop as compared to number of organoids from healthy (undamaged) gland. Intriguingly, these organoids display a cystic morphology whereas the organoids from undamaged gland are predominantly dense. Compared to the dense organoids, cystic organoids are more limited in expandability. Transcriptomic analysis revealed distinct epithelial phenotypes with cystic organoids composed of simple columnar/cuboidal epithelium and dense organoids of stratified squamous epithelium. Further transcriptional profiling showed that the cystic organoid type more closely resembles the pituitary phenotype, at least to an immature state, and that cystic organoids display specific in vitro differentiation ability. Additional characterization of the organoids exposed several facets of pituitary stem cell regulatory pathways (such as the importance of the WNT pathway) and advanced new injury-activated stem cell markers. Finally, thorough transcriptomic analyses of both pituitary (non-)stem cell populations and organoids suggested that immune/inflammatory processes are upregulated in the stem cell compartment when activated upon injury. This reaction is potentially triggered by histones presented or released by the cells that are dying through apoptosis in the transgenic pituitary injury model. Detailed examination uncovered an 'injury signature' in the stem cell population, i.e. a set of five genes upregulated after the transgenic damage and encompassing interleukin 6 (IL6). Addition of this cytokine was found to stimulate organoid formation from healthy pituitary tissue. IL6 may thus represent one of the signals that in vivo activate pituitary stem cells as occurring upon injury. Taken together, the pituitary organoid model proves to be a valuable tool in the search of stem cell-activating factors and pathways. Intriguingly, immune/inflammatory processes also appear enriched in the pituitary stem cell population during tumor growth in the gland. This finding may point to a common central mechanism underlying stem cell activation in the pituitary in different pathological conditions. Taken together, we established a novel organoid model revealing new insights into pituitary stem cell identity and regulation. This organoid model will provide an important research tool to decipher pituitary stem cell biology in both healthy and diseased gland.

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The pituitary gland represents the core of our endocrine system and governs the fundamental processes of growth, metabolism, sexual maturation, procreation and stress response. Tumors in the pituitary occur frequently, and although typically benign, cause serious morbidity due to hormone overproduction (like in Cushing’s disease and acromegaly) and to compression effects because of local expansion and invasion into the gland and beyond (leading to hypopituitarism, chronic headache and visual dysfunctions). Despite extensive research, knowledge on pituitary tumor pathogenesis is very limited.The ‘cancer stem cell’ (CSC) model posits that tumors contain a specific subpopulation of cells that drive cancer behavior and progression. In this thesis, we searched for CSC (in case of benign tumors better referred to as ‘tumor stem cells’ or TSC) in pituitary adenomas using the side population (SP) methodology. SP cells are typified by high efflux capacity and identified as Hoechstlow cells in flow cytometry. In several cancer types, the SP appears enriched in (candidate) CSC.We identified a SP in human pituitary adenomas irrespective of hormonal phenotype. This adenoma SP, as well as the further purified SP (pSP) as depleted from endothelial and immune cells, enriches for cells that express tumor-stemness markers and signaling pathways, including epithelial-mesenchymal transition (EMT)-linked factors. EMT is a cell conversion process that plays a crucial role in several steps of cancer pathogenesis (like growth, maintenance, invasion, metastasis), and that also promotes the generation and activity of tumor-driving CSC. Pituitary adenomas were found to contain self-renewing sphere-forming cells, considered a property of CSC/TSC. These sphere-initiating cells were recovered in the pSP. Because benign human pituitary adenomas do not grow in vitro and failed to expand in immunodeficient mice, the pituitary tumor cell line AtT20 was used for further study. We identified a SP also in this cell line and found it more tumorigenic than the non-SP ‘main population’. Of the two EMT-regulatory pathways tested, inhibition of C-X-C chemokine receptor type 4 (Cxcr4) signaling reduced EMT-associated cell motility in vitro as well as xenograft tumor growth in vivo, whereas activation of TGFβ had no effect. The human adenoma pSP and AtT20 SP also showed upregulated expression of the pituitary stem cell marker SOX2.Pituitaries from dopamine receptor D2 knockout (Drd2-/-) mice, bearing prolactinomas, contain more pSP, Sox2+ and colony-forming cells than wild type glands. The stem cell compartment in the gland may thus be linked to tumorigenesis and the TSC.Because resistance to therapy is one of the hallmarks of CSC/TSC, we investigated the effect of temozolomide (TMZ), a chemotherapeutic drug more and more used to treat refractory and/or recurring pituitary tumors, on the SP of pituitary tumor cells in vitro. Our first experiments show that the SP displays (some degree of) resistance to TMZ. Additional studies are now needed to gain insight into the mechanisms underlying the SP’s refractoriness.Finally, a preliminary wide-eyed genome-expression comparison of the putative CSC/TSC (as represented by the pSP) between benign pituitary adenoma and malignant melanoma and pancreatic cancer yielded some first suggestions of factors and pathways (e.g. senescence) that may underlie the benign nature of pituitary tumors. Future studies are needed including the exploration of the functional significance of these earliest findings.In conclusion, we detected a SP in pituitary tumor with TSC-associated molecular and functional characteristics. Our study adds new elements to the unravelment of pituitary tumor pathogenesis and therapy resistance, and may eventually contribute to improved clinical management by exposing novel prognostic/predictive biomarkers and therapeutic targets.

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The pituitary is the ‘master’ endocrine gland governing essential physiological processes like growth, metabolism, procreation and stress response. Because of this key position, deficient production of one or more hormones (i.e. hypopituitarism) prominently impacts on body physiology, leading to chronic morbidity with increased mortality risk. In most cases, hypopituitarism is caused by damage suffered during life. Tumor growth in the gland can compress the healthy tissue, and pituitary tumor irradiation or resection causes tissue destruction. Moreover, hypopituitarism is a common consequence of traumatic brain injury (as e.g. caused by traffic accidents, falls, sports, violence). On the other hand, hypopituitarism can be congenital, involving mutations in genes with essential role in pituitary embryogenesis. Current treatment of pituitary deficiency consists of multiple-hormone replacement therapy. This medication is suboptimal given the inadequacy to reproduce the pituitary’s natural pulsatile secretion pattern. In addition, the administered hormones cause burdening endocrine and cardiovascular side effects. Regenerating the defective pituitary tissue and function would provide an interesting therapeutic alternative. For that purpose, a better understanding of pituitary regeneration and hypopituitarism is necessary. In the first part of this thesis (Chapter 3), we exploited our recently developed transgenic mouse model of damage-induced hypopituitarism to further unravel regenerative parameters and mechanisms of the gland, eventually instrumental to restore defective pituitary tissue. In a second part (Chapter 4), we prepared the development of in vitro human hypopituitarism models by testing pituitary differentiation protocols on pluripotent embryonic stem cells (ESC) and by generating hypopituitarism patient-derived induced pluripotent stem cells (iPSC).Previously, our group developed a mouse hypopituitarism model by transgenically inducing damage in the gland. In this ‘GHCre/iDTR’ model in which somatotropes (cells producing growth hormone, GH) are destroyed after treatment with diphtheria toxin (DT), substantial regeneration (up to 60%) of the somatotropes was observed 4-6 months after their destruction. Stem cells are most likely involved in this regeneration process, as indicated by their prompt expansion and emergence of GH expression in these cells.In the first part of the thesis (Chapter 3), we characterized the regenerative process of the pituitary in more depth, in particular focusing on molecular and time-related aspects. First, extending the recovery period up to 19 months did not result in higher regeneration levels. Remarkably, the pituitary’s regenerative competence was found to quickly disappear with age. Middle-aged (8-months old) mice did not show any recovery anymore of the affected somatotrope cell population. Further surprisingly, prolonging the DT treatment period (in young-adult mice) from 3 days (3DT) to 10 days (10DT) annihilated the regenerative capacity of the gland although the initial somatotrope ablation grade remained similar (80-90%). The stem cell compartment still expanded promptly after 10DT and retained intrinsic stem-cell functionality as probed by sphere formation and differentiation capacity. To search for potential molecular grounds underlying this reparative failure, the stem cell-clustering ‘side population’ (SC-SP) of the non-regenerating (10DT) pituitary was compared to the SC-SP of the regenerating (3DT) pituitary using whole-genome expression analysis. A number of stemness and embryonic factors and pathways were found lower expressed in the SC-SP of the non-regenerating pituitary. Taken together, the regenerative capacity of the pituitary is limited both in age-related terms and final efficacy, and likely relies on stem cell-associated pathway activation. Dissection of the molecular profiles may eventually identify targets to induce or boost regeneration in situations of (injury-related) pituitary deficiency.In the second part of the thesis (Chapter 4), we aimed at developing an in vitro pituitary (disease) model for humans. Knowledge on genetic hypopituitarism is poor and only derived from mouse models. It is not known whether these principles also apply to humans. Here, we started to test and optimize two protocols recently described to induce the development of pituitary hormonal cells from ESC. In the first (3D) protocol, embryoid bodies (EB) were generated from human ESC (hESC) which had first been standardly expanded on mouse fibroblast feeder layers. Treatment of the EB with a SHH agonist resulted in upregulation of pituitary specification markers. However, further hormonal differentiation was not achieved and reproducibility was low, likely due to the heterogeneous composition of the EB containing residual feeder cells. Therefore, we turned to a second (2D) approach in which feeder cells were first depleted by growing the hESC on Matrigel. Transient treatment of the purified hESC with a BMP inhibitor under continuous blockage of TGFβ signaling induced pre-placodal specification. Subsequent pulse activation of the SHH pathway and culture in neurobasal medium stimulated the development of ACTH-expressing corticotropes in the hESC culture. Inhibition of the NOTCH pathway resulted in the appearance of GH-expressing cells in the cultures. Parallel to these tests, we generated iPSC from somatic cells (skin fibroblasts) of a PIT1-mutant hypopituitarism patient, and validated three iPSC lines for pluripotency and karyotype. Two lines were found valid and will be useful to create an in vitro human hypopituitarism model. Taken together, our in vitro models will eventually be valuable to decipher molecular mechanisms of normal and deficient development of the pituitary in humans, and to evaluate potential drugs for treatment and/or repair of hypopituitarism.In conclusion, our study more deeply analyzed the pituitary regenerative process after cell-ablation injury and observed negative influences of age and duration of infliction. In addition, we identified some molecular embryonic/stemness pathways that may underlie the stem cells’ regenerative response. Finally, we started to create conditions to allow developing in vitro human pituitary (disease) models for studying human pituitary embryogenesis, biology and hypopituitarism. The identification of cellular and molecular players in pituitary regeneration and pituitary disease may eventually lead to targets and strategies for restoration and/or regeneration at the tissue and cellular level that could provide a more favorable clinical outcome for hypopituitarism than exogenous hormone replacement.

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