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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 gland takes, together with the hypothalamus, the main lead in coordinating the endocrine system to regulate vital physiological body processes like growth, puberty, reproduction and stress response. Because of this central role, malfunctioning of the pituitary can cause severe disorders like diabetes, cardiovascular disease, osteoporosis, infertility and psychological problems. Pituitary hormonal cell populations must therefore be maintained in a controlled and balanced manner. Previous studies revealed the existence of stem cells in the pituitary gland, mainly based on a side population (SP) and SOX2-expressing phenotype. Their role, however, remains uncertain. Lineage tracing showed that SOX2+ cells can give rise to all pituitary endocrine cell types during postnatal homeostasis, although only at a low degree. After damage in the gland, the pituitary stem cells are activated and appear involved in the subsequent regenerative process. Recently, it was found that the pituitary SP and SOX2+ stem cells are activated and expanded during tumor development and growth in the gland as occurring in the ‘dopamine receptor D2 knockout (Drd2-/-) mouse. Tumorigenesis is the most common pathology of the pituitary. However, underlying mechanisms remain elusive and current therapies are often unsatisfactory because of inoperability or therapy resistance.In the first part of our study, we investigated the role and significance of the pituitary stem cells in the postnatal gland by depleting the SOX2+ cells through diphtheria toxin (DT)-mediated ablation. In the second part, we further characterized the stem cells in the tumorous Drd2-/- pituitary in search of their role in development, progression and therapy resistance of pituitary tumors.Administration of DT to adult Sox2CreERT2/+;R26iDTR/+ mice (after tamoxifen-induced expression of DT receptor or DTR in SOX2+ cells) resulted in 80% obliteration of SOX2+ cells in the endocrine pituitary, coinciding with reduced pituisphere-forming activity. Counterintuitively for a stem cell population, the SOX2+ cell compartment did not repopulate. Considering the more active phenotype of the stem cells during early-postnatal pituitary maturation, SOX2+ cell ablation was also performed in 4- and 1-week-old animals. Ablation grade diminished with decreasing age and was accompanied by a proliferative reaction of the SOX2+ cells, suggesting a rescue attempt. Despite this activation, SOX2+ cells did also not recover. Finally, the major SOX2+ cell depletion in adult mice did not affect the homeostatic maintenance of pituitary hormonal cell populations, neither the cell (corticotrope) remodeling response to adrenalectomy challenge.To characterize the pituitary stem cell population during tumorigenesis, whole-genome expression analysis using RNA-sequencing (RNA-seq) was performed. Analysis revealed upregulation of different stemness factors and pathways including WNT, epithelial-mesenchymal transition (EMT), chemokine signaling and NOTCH in the SP of Drd2-/- pituitary. Transgenic lineage tracing of SOX2+ cells before and during pituitary tumor growth in the Drd2-/- mouse showed that the pituitary stem cells do not, or at least not to a substantial level, give rise to the tumor cells, suggesting rather an indirect, potentially paracrine role. Finally, higher resistance to the chemotherapeutic drug temozolomide was observed in the stem cell fraction of both mouse and human pituitary tumors as analyzed in vitro.In summary, our study shows that pituitary SOX2+ cells fail to regenerate after major depletion which does not affect adult endocrine cell homeostasis and remodeling. Thus, pituitary SOX2+ cells may constitute a copious stem cell reserve or may have other critical role(s) still to be clearly defined. In addition, pituitary stem cells appear activated during tumor formation in the (Drd2-/-) pituitary regarding their stemness molecular phenotype but are not directly linked to the tumor cells. This activation may imply a reaction against tumor development in the gland which may then indirectly affect tumor growth by paracrine signaling. Moreover, the stem cell fraction in pituitary tumors appears more resistant to chemotherapy; further characterization may lead to insights in the underlying mechanisms of therapy resistance and eventually to new therapeutic opportunities.In conclusion, our study adds new elements to the role of pituitary stem cells during homeostasis and tumorigenesis, but more research is clearly needed.