Generation of mouse hepatobiliary organoids from hepatocyte progenitors and cholangiocytes isolated from healthy adult mouse liver

Number of liver organoids have been reported, though it is not clearly shown whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors called small hepatocytes (SHs) and cholangiocytes. SHs differentiate and form the bile canalicular network in HBTOs and secret metabolites into the canaliculi, which are then transported into the biliary structure. Hepatocytes in the organoid acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. We provide the step-by-step protocol for induction of HBTO including isolation of cholangiocytes and SHs and co-culture of these two types of cell to generate functional connections between hepatocytes and cholangiocytes.

Discovery of lipotoxicity-sensitive transcription factors in non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease is continuum of disorders among which non-alcoholic steatohepatitis (NASH) is particularly associated with a negative prognosis. Hepatocyte lipotoxicity is one of the main pathogenic factors of liver fibrosis and NASH. However, the molecular mechanisms regulating this process are poorly understood. Here, we integrated transcriptomic and chromatin accessibility analyses from human liver and mouse hepatocytes to identify lipotoxicity-sensitive transcription factors. We found that the transcription factors MAFK and TCF4 were activated in liver from NASH patients and by mouse hepatocyte lipotoxicity. Genetic deletion of these transcription factors protected hepatocytes against saturated fatty acid oversupply. Notably, MAFK- and TCF4-regulated gene expression linked to lipotoxicity closely correlated with transcriptional patters in fibrosis progression in NASH patients. Collectively, our findings uncovered novel molecular insights into lipotoxicity-induced NASH, revealing the relevance and therapeutic potential of MAFK and TCF4 in human disease.

Generation of functional liver tissue by establishing hepatobiliary connections ex vivo

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. We have developed a hepatobiliary organoid using mouse hepatocyte progenitors and cholangiocytes. Hepatocyte metabolites were secreted into the bile canaliculi, and then transported into the biliary structure. Hepatocytes in the organoid acquired and maintained metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we established functional liver tissue incorporating a bile drainage system ex vivo. This hepatobiliary organoid enabled us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

Effect of Sex Specific differences on Function of Induced Hepatocyte-Like Cells Generated from Male and Female Mouse Embryonic Fibroblasts

Background The liver is one of the vital organ involve in detoxification and metabolism. The sex based differences between the functionality of male and female liver has been previously reported i.e. male’s liver are good in alcohol clearance and lipid metabolism, while, female’s liver are better in cholesterol metabolisms. To date, studies on novel drug toxicity have not considered the sex specific dimorphic nature of the liver. However, the use of hepatocyte like cells to treat liver diseases has increased recently.Methods Mouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEF) were isolated from back skin tissue of each embryo. MEF were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using lentiviral system. The transduced MEF were further treated with hepatocyte conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays and finally Whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCL4 induced acute liver mouse model.Results In this study, we evaluated the sex specific effect of mouse hepatocyte-like cells (miHep) induced from male and female specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus, and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex.Conclusion In conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

Primary mouse hepatocyte isolation

This protocol is intended to isolate primary hepatocytes from mus musculus (mouse) for the purposes of culturing primary hepatocytes. Hepatocytes can be used for approximately 5 days from the time of isolation, and cannot be frozen for future use. A single isolation from a single mouse, including time to prepare reagents, takes approximately 2 hours.