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Image by Louis Reed

Article by Ben Yu, François Chesnais & Margaretha Morsink

Engineered Liver Organoids for Bile Duct Development and Disease Modeling

Bile duct disorders are medical conditions that disrupt the normal function of bile ducts to carry bile from the liver and gallbladder to the small intestine, which can lead to the destruction of liver tissue and have potentially fatal complications. Bile duct disorders are hard to study because the development of bile ducts, including their formation and interaction with surrounding blood vessels in liver development, is not well understood and lacks precise human models for further investigation. This makes finding effective treatments also difficult due to the inability to replicate the bile duct environment in laboratory settings. This study addresses these challenges by constructing a 3D co-culture system that integrates human-induced pluripotent stem cell (hiPSC)-derived liver progenitors with artificial blood vessels. Using this system, this group of researchers was able to successfully model the formation of bile ducts and their interaction with blood vessels, allowing for a better understanding of bile duct diseases such as Alagille syndrome and cholestasis.

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What did these researchers do?

In this study, researchers developed a 3D co-culture system that could model the formation of bile ducts and their interaction with blood vessels in liver development. They mixed hiPSC liver progenitor cells with artificial blood vessels composed of hiPSC-derived smooth muscle and endothelial cells. Over three weeks of culture, these liver progenitors differentiated into functional bile duct cells showing epithelial characteristics, including intercellular junctions, secretory function, and apical microvilli. These characteristics demonstrated that the bile ducts were functioning properly and were mimicking the structural properties of real bile ducts. Moreover, the authors analyzed key cellular signaling pathways, such as TGFβ and Notch signaling, which are key molecular mechanisms underlying bile duct formation. Transplantation of these organoids onto the livers of cholestatic mice - mice that had impaired flow of bile from the liver to the intestine - temporarily relieved symptoms of liver injury. This demonstrated their potential for studying biliary diseases, genetic disorders, and applications in regenerative medicine. From these results, the researchers concluded that their model effectively mimics key processes of bile duct development and could serve as a powerful tool for studying biliary diseases and testing therapeutic approaches for cholestatic and congenital biliary disorders.

 

Why is this important?

Many bile duct diseases, such as cholestasis and Alagille syndrome, are still challenging to study and treat because the current models cannot accurately mimic human liver development. Traditional 2D cell cultures cannot reproduce the three-dimensional structure and interactions between bile ducts and blood vessels, while animal models often fail to capture human-specific disease mechanisms. This study is groundbreaking because it goes beyond past approaches in creating a 3D co-culture system using hiPSC-derived liver progenitors and artificial blood vessels that successfully replicate human bile duct formation and its interaction with blood vessels. Although researchers mostly used healthy hiPSC-derived cells to model bile duct development, they also used JAG1-knockout hiPSCs to imitate genetic disorders and disease conditions like Alagille syndrome. Compared to previous models, the current system allows for the study of critical signaling pathways such as TGFβ and Notch that drive the development of bile ducts and provides a new perspective for understanding the molecular basis of genetic disorders. The ability to test the system in a cholestatic mouse model further confirms its utility in preclinical therapeutic applications, making this a great improvement compared to existing models. Ultimately, this development paves the way for more effective drug testing, disease modeling, and the development of regenerative medical approaches for biliary diseases.

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How did the researchers do this?

To construct the 3D co-culture system, researchers first differentiated hiPSCs into liver progenitor cells, smooth muscle cells (SMCs), and endothelial cells (ECs) following established protocols. Then, they formed artificial blood vessels by encapsulating the SMCs in a tube-like collagen scaffold while subsequently lining the lumen of the scaffold with ECs. The liver progenitor cells were cultured into bile duct organoids in a separate manner. These were differentiated through different markers, as the liver progenitor cells were marked by HNF4a and the bile duct structures were marked with CK19.

 

The researchers then integrated the organoids with the synthetic blood vessels by wrapping the liver progenitors around the tube-like structures and embedding the system in a matrix containing extracellular proteins such as laminin. This was maintained for three weeks, promoting cellular interactions and maturation, during which the liver progenitors matured into function bile ducts displaying intercellular junctions, secretory function, and apical microvilli. Two signaling pathways that are critical for the formation of bile ducts are the TGFβ and Notch signaling pathways. In order to study the molecular mechanisms driving the development of the bile duct, researchers studied these pathways using used single-cell RNA sequencing and genetic knockout of essential genes like JAG1.

 

Finally, the researchers assessed the structural and functional features of the bile ducts developed in the 3D co-culture system using a variety of different techniques. Immunofluorescence microscopy allowed for the identification of important epithelial marker expressions such as CK19, CK7, ZO-1, and F-actin, which validated proper bile duct formation. Meanwhile, transmission electron microscopy (TEM) provided high-resolution images of microvilli and tight junctions, further confirming proper epithelial differentiation.

 

To evaluate the system in vivo, they transplanted the organoids onto cholestatic mice and monitored symptoms like jaundice and bilirubin levels. Histological and immunofluorescence analyses confirmed host-graft integration, formation of functional luminal connections, and temporary improvement of liver function. Unfortunately, the transplantation only increased lifespan by approximately three days, as the bile ducts could not fully integrate with the host’s biliary system over the long term. This indicates that although engineered bile ducts could mimic real structures and functions, further refinement of the system is needed to support long-term integration and functionality.

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Formation of 3D liver organoid with artificial blood vessels for bile duct modeling

What comes next?

While great advancements have been made in the development of fully functional and mature bile ducts through the 3D co-culture system, significant limitations still remain. One major challenge is the incomplete maturation of the engineered bile ducts, as evidenced by a lack of primary cilia. Additionally, the therapeutic effects of the transplanted bile ducts were temporary, improving survival in cholestatic mice by only a few days. Most importantly, the study’s transplantation experiment was conducted exclusively on mice, which means that further testing is required before considering applications to human patients.

 

Overall, the ultimate goal of this study is to create engineered bile duct tissues for potential human transplantation and to develop a reliable model for studying diseases related to the bile system. Future studies should aim to optimize the co-culture method to achieve complete bile duct maturation and long-term functionality. This includes ensuring complete integration of a fully hepatobiliary network that connects the engineered bile ducts to the liver’s natural bile system. By addressing these challenges, this platform can connect the gap between laboratory models and clinical treatments for biliary diseases.

© 2024 by Tissue Engineering Resource Center 

With support from the NIBIB.

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