A decade of liver organoids: Advances in disease modeling.

Liver organoids are three-dimensional cellular tissue models in which cells interact to form unique structures in culture. During the past 10 years, liver organoids with various cellular compositions, structural features, and functional properties have been described. Methods to create these advanced human cell models range from simple tissue culture techniques to complex bioengineering approaches. Liver organoid culture platforms have been used in various research fields, from modeling liver diseases to regenerative therapy. This review discusses how liver organoids are used to model disease, including hereditary liver diseases, primary liver cancer, viral hepatitis, and nonalcoholic fatty liver disease. Specifically, we focus on studies that used either of two widely adopted approaches: differentiation from pluripotent stem cells or epithelial organoids cultured from patient tissues. These approaches have enabled the generation of advanced human liver models and, more importantly, the establishment of patient-tailored models for evaluating disease phenotypes and therapeutic responses at the individual level.

Quantification of Proliferative and Dead Cells in Enteroids.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or "enteroids". Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2'-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis.

Intestinal barrier function is required for the maintenance of mucosal homeostasis. Barrier dysfunction is thought to promote progression of both intestinal and systemic diseases. In many cases, this barrier loss reflects increased permeability of the paracellular tight junction as a consequence of myosin light chain kinase (MLCK) activation and myosin II regulatory light chain (MLC) phosphorylation. Although some details about MLCK activation remain to be defined, it is clear that this triggers perijunctional actomyosin ring (PAMR) contraction that leads to molecular reorganization of tight junction structure and composition, including occludin endocytosis. In disease states, this process can be triggered by pro-inflammatory cytokines including tumor necrosis factor-α (TNF), interleukin-1ß (IL-1ß), and several related molecules. Of these, TNF has been studied in the greatest detail and is known to activate long MLCK transcription, expression, enzymatic activity, and recruitment to the PAMR. Unfortunately, toxicities associated with inhibition of MLCK expression or enzymatic activity make these unsuitable as therapeutic targets. Recent work has, however, identified a small molecule that prevents MLCK1 recruitment to the PAMR without inhibiting enzymatic function. This small molecule, termed Divertin, restores barrier function after TNF-induced barrier loss and prevents disease progression in experimental chronic inflammatory bowel disease.