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Dissecting the role of biliary epithelial cells during non-alcoholic fatty liver disease progression

Ece Yildiz

The liver is the largest solid organ and the only one capable of using regenerative mechanisms to recover its mass fully. Although liver regeneration from acute injuries has been effective and extensively studied, chronic liver damage has adverse effects on hepatic histology, such as fibrosis or cirrhosis, suggesting that regenerative mechanisms have been disrupted. The most common form of chronic liver disorder, non-alcoholic fatty liver disease (NAFLD), is a growing cause of end-stage liver disease worldwide and is estimated to increase 2-3-fold by 2030. Despite the enormous amount of ongoing research on this topic, no treatment is available, leaving liver transplantation as the only current option. One approach to revert this situation focuses on facultative liver stem cells derived from the biliary epithelium. This recent alternative comes from the breakthrough that biliary epithelial cells (BEC) can be assembled into complex three-dimensional organoid structures in vitro from bile duct-derived stem cells. These BEC-organoids can be expanded as stem cells, with a similar phenotype to injury-induced BECs in vivo, and also can be differentiated into functional hepatocyte-like cells with engraftment ability upon transplantation. Considering the potential of BEC-organoids, I focused on dissecting the role and unveiling the potential of BECs in the context of NAFLD. BECs undergo rapid reprogramming and proliferation in chronic liver diseases, including the fibrotic stage of NAFLD, a process known as ductular reaction (DR). Thus, studying DR provides new insights into the BEC expansion mechanisms. For this purpose, I first investigated the effect of hepatic overload on BECs to study the initiation of DR during the early stages of NAFLD. I demonstrated that lipid overload induces the conversion of adult cholangiocytes into proliferating BECs and promotes their expansion via the activation of the E2F transcription factors, which drives cell cycle progression while promoting glycolytic metabolism. These observations, while correlative, reveal unexpected connections between lipid metabolism and stemness, and set the ground for future research in understanding the role and the therapeutic potential of lipid metabolism and E2Fs in controlling BEC activation. On the other hand, DR correlates closely with the severity of fibrosis in NAFLD, even though the function is largely unknown. Thus, I studied the effect of the mechanical properties of the fibrotic environment in BEC-organoid cultures by using the synthetic matrix we developed together with colleagues. The investigation of BECs cultured in a defined mechanical environment mimicking fibrotic stiffness revealed decreased stem cell capacities and increased inflammation, hepatic injury, and matrix metalloproteases. Finally, by using recently published data comparing BEC-organoid cultures from healthy and NASH patients, I revealed that hydrogels with fibrotic stiffness mimic the phenotype of organoids derived from NASH patients. As a result, investigating aberrant stiffness will enable the development of powerful DR models and future therapeutics for enhancing stem cell-mediated liver regeneration. Overall, the results from this thesis should spark future enthusiasm for the potential of BECs in studying DR regenerative mechanisms and developing therapies.

EPFL2022

Engineering Modular 3D Liver Culture Microenvironments In Vitro to Parse the Interplay between Biophysical and Biochemical Microenvironment Cues on Hepatic Phenotypes

Ece Yildiz

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influences phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHHs) cultured in a commercially available perfused bioreactor. A 3D printing-based alginate microwell system is designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver are engineered to replace polystyrene scaffolds, and the biochemical microenvironment is modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increases tissue density, albumin secretion, and CYP3A4 activity but also upregulates inflammatory markers. Basal inflammatory markers are lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibit the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA-Seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

WILEY-V C H VERLAG GMBH2022

Bile Acids Signal via TGR5 to Activate Intestinal Stem Cells and Epithelial Regeneration

Maroun Bou Sleiman, Gaby El Alam, Alessia Perino, Kristina Schoonjans, Giovanni Sorrentino, Ece Yildiz

BACKGROUND & AIMS: Renewal and patterning of the intestinal epithelium is coordinated by intestinal stem cells (ISCs); dietary and metabolic factors provide signals to the niche that control ISC activity. Bile acids (BAs), metabolites in the gut, signal nutrient availability by activating the G protein-coupled bile acid receptor 1 (GPBAR1, also called TGR5). TGR5 is expressed in the intestinal epithelium, but it is not clear how its activation affects ISCs and regeneration of the intestinal epithelium. We studied the role of BAs and TGR5 in intestinal renewal, and regulation of ISC function in mice and intestinal organoids. METHODS: We derived intestinal organoids from wild-type mice and Tgr5(-/-) mice, incubated them with BAs or the TGR5 agonist INT-777, and monitored ISC function by morphologic analyses and colony-forming assays. We disrupted Tgr5 specifically in Lgr5-positive ISCs in mice (Tgr5ISC(-/-) mice) and analyzed ISC number, proliferation, and differentiation by flow cytometry, immunofluorescence, and organoid assays. Tgr5ISC(-/-) mice were given cholecystokinin; we measured the effects of BA release into the intestinal lumen and on cell renewal. We induced colitis in Tgr5ISC(-/-) mice by administration of dextran sulfate sodium; disease severity was determined based on body weight, colon length, and histopathology analysis of colon biopsies. RESULTS: BAs and TGR5 agonists promoted growth of intestinal organoids. Administration of cholecystokinin to mice resulted in acute release of BAs into the intestinal lumen and increased proliferation of the intestinal epithelium. BAs and Tgr5 expression in ISCs were required for homeostatic intestinal epithelial renewal and fatespecification, and for regeneration after colitis induction. Tgr5ISC(-/-) mice developed more severe colitis than mice without Tgr5 disruption in ISCs. ISCs incubated with INT-777 increased activation of yes-associated protein 1 (YAP1) and of its upstream regulator SRC. Inhibitors of YAP1 and SRC prevented organoid growth induced by TGR5 activation. CONCLUSIONS: BAs promote regeneration of the intestinal epithelium via activation of TGR5 in ISCs, resulting in activation of SRC and YAP and activation of their target genes. Release of endogenous BAs in the intestinal lumen is sufficient to promote ISC renewal and drives regeneration in response to injury.

2020