Pituitary deficiency and regeneration: in search of underlying mechanisms and therapeutic targets with focus on Wnt/Lgr5

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.