Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees.

BACKGROUND AND AIMS: Globally, liver diseases account for 2 million deaths per year. For those with advanced liver disease the only curative approach is liver transplantation. However, less than 10% of those in need get a liver transplant due to limited organ availability. To circumvent this challenge, there has been a great focus in generating a bioengineered liver. Despite its essential role in liver functions, a functional biliary system has not yet been developed. In this framework, exploration of epithelial cell self-organogenesis and microengineering-driven geometrical cell confinement allow to envision the bioengineering of a functional biomimetic intrahepatic biliary tract. APPROACH: three-dimensional (3D) bile ducts were built in vitro by restricting cell adhesion to two-dimensional (2D) patterns to guide cell self-organization. Tree shapes mimicking the configuration of the human biliary system were micropatterned on glass slides, restricting cell attachment to these areas. Different tree geometries and culture conditions were explored to stimulate self-organogenesis of normal rat cholangiocytes (NRCs) used as a biliary cell model, either alone or in co-culture with human umbilical endothelial cells (HUVECs). RESULTS: Pre-seeding the micropatterns with HUVECs promoted luminogenesis with higher efficiency to yield functional branched biliary tubes. Lumen formation, apico-basal polarity, and preservation of the cholangiocyte phenotype were confirmed. Moreover, intact and functional biliary structures were detached from the micropatterns for further manipulation. CONCLUSION: This study presents physiologically relevant 3D biliary duct networks built in vitro from 2D micropatterns. This opens opportunities for investigating bile duct organogenesis, physiopathology, and drug testing.

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Biophysical Control of Bile Duct Epithelial Morphogenesis in Natural and Synthetic Scaffolds.

The integration of bile duct epithelial cells (cholangiocytes) in artificial liver culture systems is important in order to generate more physiologically relevant liver models. Understanding the role of the cellular microenvironment on differentiation, physiology, and organogenesis of cholangiocytes into functional biliary tubes is essential for the development of new liver therapies, notably in the field of cholangiophaties. In this study, we investigated the role of natural or synthetic scaffolds on cholangiocytes cyst growth, lumen formation and polarization. We demonstrated that cholangiocyte cyst formation efficiency can be similar between natural and synthetic matrices provided that the mechanical properties of the hydrogels are matched. When using synthetic matrices, we also tried to understand the impact of elasticity, matrix metalloprotease-mediated degradation and integrin ligand density on cyst morphogenesis. We demonstrated that hydrogel stiffness regulates cyst formation. We found that controlling integrin ligand density was key in the establishment of large polarized cysts of cholangiocytes. The mechanism of lumen formation was found to rely on cell self-organization and proliferation. The formed cholangiocyte organoids showed a good MDR1 (multi drug resistance protein) transport activity. Our study highlights the advantages of fully synthetic scaffold as a tool to develop bile duct models.

Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Renin is an aspartyl protease essential for the control of blood pressure and was long suspected to have cellular receptors. We report the expression cloning of the human renin receptor complementary DNA encoding a 350-amino acid protein with a single transmembrane domain and no homology with any known membrane protein. Transfected cells stably expressing the receptor showed renin- and prorenin-specific binding. The binding of renin induced a fourfold increase of the catalytic efficiency of angiotensinogen conversion to angiotensin I and induced an intracellular signal with phosphorylation of serine and tyrosine residues associated to an activation of MAP kinases ERK1 and ERK2. High levels of the receptor mRNA are detected in the heart, brain, placenta, and lower levels in the kidney and liver. By confocal microscopy the receptor is localized in the mesangium of glomeruli and in the subendothelium of coronary and kidney artery, associated to smooth muscle cells and colocalized with renin. The renin receptor is the first described for an aspartyl protease. This discovery emphasizes the role of the cell surface in angiotensin II generation and opens new perspectives on the tissue renin-angiotensin system and on renin effects independent of angiotensin II.