Intrahepatic cholangiocyte organoids for bile-duct tissue engineering and disease modeling

Abstract

The biliary tract is structurally and functionally a highly complex system in the human body, and is composed of the intra- and extrahepatic bile duct and gallbladder. Bile is initially produced by hepatocytes, secreted into the bile canaliculi and then flows through the bile ducts, which deliver bile to the gallbladder and finally to the duodenum. Cholangiocytes are epithelial cells that line the biliary tree, which constitutes only 3-5% of the total cell mass of the liver, but they play a crucial role in the final bile composition by regulating transmembrane transport proteins. These transporters are located on the apical or basolateral plasma membranes and include channels, efflux pumps and exchangers. Impairment of these transporters could lead to inflammation, cholestasis, fibrosis and liver injury, finally causing liver failure. Liver transplantation is the only therapeutic strategy for this, which presents a significant health care and economic burden. To investigate the molecular and cellular mechanisms of cholangiopathies and to develop therapeutic strategies, suitable models are needed. This thesis aimed to develop a bioengineered bile duct that can recapitulate key structures and functions of cholangiocytes for applications in biliary pathophysiological research.Various cell types have been proposed as potential cell source for producing cholangiocyte-like cells (CLCs). Chapter 2 gives an elaborative overview of the current understanding of tissue-engineered bile ducts and the propagation of mature functional cholangiocytes. In Chapter 3, we successfully established an efficient, stepwise method for differentiating human intrahepatic cholangiocyte organoids (ICOs) into functional CLCs that recapitulate key characteristics. Human ICO-derived CLCs showed key characteristics of cholangiocytes, including expression of structural and functional markers. Furthermore, human ICO-derived CLCs applied on HFM to form bioengineered bile ducts, will facilitate studying cholangiopathies and allow for developing therapeutic strategies. In addition, we pioneered in applications for CLCs to investigate drug-induced bile duct injury in Chapter 4. Drug-induced bile duct injury is a frequent clinical problem with variable pathological phenotypes of which the mechanisms of the bile duct damage are still poorly understood. To uncover drug-induced bile duct injury, we developed an in vitro model suitable for mimicking drug-induced bile duct injury under cholestatic or non-cholestatic conditions in Chapter 4. Despite significant advances in the field of in vitro propagation of cholangiocytes and cholangiopathy studies, several challenges remain including reducing the large variations observed in organoid cultures. One way to circumvent this is to generate cholangiocyte cultures without the use of animal-derived materials. To tackle this issue, in Chapter 5 our final objective was to progress towards the development of a chemically defined culture system that could be employed in regenerative medicine and would be suitable for creating implantable cholangiocytes. Finally, we provide a summary and discussion in Chapter 6, ending with future perspectives of the ICOs as important resources for bile duct tissue engineering and disease modeling.