Induction of Steatohepatitis and Liver Tumorigenesis by Enforced Snail Expression in Hepatocytes.

Snail is a transcription factor that regulates many cellular events involved in development, homeostasis, and disease. In hepatocellular carcinoma (HCC), Snail induces epithelial-to-mesenchymal transition that confers invasive properties on tumor cells during HCC progression and malignancy. Snail activation observed in HCC mouse models suggests its involvement not only in progression, but also onset of HCC. However, it remains unclear whether Snail directly contributes to HCC initiation or whether it supports HCC initiation promoted by other oncogenes. In this study, we generated mouse models for liver-specific and hepatocyte-specific overexpression of Snail to show the independent roles of Snail in liver homeostasis and disease. Enforced Snail expression resulted in liver and hepatocyte enlargement, inflammatory cell infiltration in the liver, lipid accumulation in hepatocytes, substantial increases in serum alanine aminotransferase and bile acids, yellow discoloration of tissues caused by bilirubin accumulation, and liver tumorigenesis. Snail overexpression suppressed mRNA expression of the tight junction components claudins and occludin and that of proteins associated with bile acid metabolism, leading to disruption of the biliary canaliculus formed among hepatocytes and excretion of abnormal amounts of unusual bile acids from hepatocytes. In conclusion, enforced Snail expression in hepatocytes is sufficient for induction of steatohepatitis and liver tumorigenesis through disruption of the biliary canaliculus and bile acid homeostasis in the liver.

Brief summary of the current protocols for generating intestinal organoids.

The intestine has fundamental functions for the maintenance of homeostasis, including food digestion and nutrient/water absorption. Although the lumen of the intestine is always exposed to pathogens, intestinal epithelial cells form monolayer sheets that act as an epithelial barrier to prevent the invasion of pathogens. Thus, disruption of the intestinal epithelial barrier causes inflammatory bowel diseases. To investigate the details of these intractable intestinal diseases, it is necessary to analyze the characteristics of intestinal epithelial cells in vitro. However, it is difficult to maintain and propagate intestinal epithelial cells in culture. Recently, intestinal organoid culture systems have been established, in which differentiated intestinal epithelial lineage cells can be continuously produced from intestinal stem cells and form epithelial organoids with crypt-like structures in long-term culture. Moreover, intestinal epithelial organoids can be generated not only from intestinal tissue-derived cells, embryonic stem cells, and induced pluripotent stem cells, but also by inducing direct conversion of nonintestinal somatic cells into intestinal epithelial cells. These intestinal organoids can be used in basic studies for understanding the mechanisms underlying intestinal development and diseases and will be applied in future transplantation therapy and drug discovery to treat intestinal diseases.

Suppression of lethal-7b and miR-125a/b Maturation by Lin28b Enables Maintenance of Stem Cell Properties in Hepatoblasts.

UNLABELLED: In liver development, hepatoblasts that act as hepatic stem/progenitor cells proliferate and differentiate into both hepatocytes and cholangiocytes to form liver tissues. Although numerous factors contribute to this event, little is known about the roles of microRNAs in hepatoblast proliferation and differentiation. In this study, we focused on the lineage-28 (Lin28) family proteins, which are required for microRNA regulation in pluripotent stem cells and cancer cells, and investigated their roles as regulatory factors for the properties of hepatoblasts. CONCLUSION: Lin28b was specifically expressed in hepatoblasts, and its suppression induced growth arrest and cholangiocyte differentiation of hepatoblasts; mechanistically, Lin28b positively regulates the expression of Lin28b itself and cell cycle-related proteins in hepatoblasts by suppressing the maturation of target microRNAs, lethal-7b and miR-125a/b, enabling maintenance of the stem cell properties of hepatoblasts, such as their capabilities for proliferation and bi-lineage differentiation, during liver development. (Hepatology 2016;64:245-260).

Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture.

Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.

Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature.

Direct reprogramming, inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors, is a technology with anticipated medical applications. However, due to the many unresolved aspects of the induction mechanisms, it is essential to thoroughly analyze the epigenomic state of the generated cells. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs. We found that the CpG methylation state was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, leading to the medical applications of this technology.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Direct Lineage Reprogramming of Mouse Fibroblasts to Acquire the Identity of Fetal Intestine-Derived Progenitor Cells.

Intestinal organoids are useful models for studying the characteristics of intestinal diseases and their treatment. However, a major limiting factor in their usability is the need for donor tissue fragments or pluripotent stem cells to generate the organoids. Here, we describe an approach to generate intestinal organoids from fibroblasts, a new source. We used direct reprogramming technology to generate cells with the properties of fetal intestine-derived progenitor cells (FIPCs) from mouse embryonic fibroblasts (MEFs). These induced FIPCs (iFIPCs) can give rise to cells resembling intestinal stem cells (ISCs), henceforth referred to as induced ISCs (iISCs). These iFIPCs and iISCs form spherical and budding organoids, respectively, similar to FIPCs and ISCs. These induced intestinal organoids could be used for studies on intestinal diseases and regenerative therapy.

Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells.

Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytes and cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.

Cell Aggregation Culture Induces Functional Differentiation of Induced Hepatocyte-like Cells through Activation of Hippo Signaling.

Recent progress in direct lineage reprogramming has enabled the generation of induced hepatocyte-like (iHep) cells and revealed their potential as an alternative to hepatocytes for medical applications. However, the hepatic functions of iHep cells are insufficient compared with those of primary hepatocytes. Here, we show that cell-aggregate formation can rapidly induce growth arrest and hepatic maturation of iHep cells through activation of Hippo signaling. During formation of iHep cell aggregates, Yap inactivation is induced by actin reorganization and intercellular adhesion, leading to upregulation of Hnf1α expression in the absence of the Yap/Tead/Chd4 transcriptional repressor unit. Hnf1α then acts as a central transcription factor that regulates liver-enriched gene expression in iHep cell aggregates and induces functional differentiation of iHep cells. Moreover, iHep cell aggregates efficiently reconstitute injured liver tissues and support hepatic function after transplantation. Thus, iHep cell aggregates may provide insights into basic research and potential therapies for liver diseases.

Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming.

Intestinal organoids hold great promise as a valuable tool for studying and treating intestinal diseases. The currently available sources of human intestinal organoids, tissue fragments or pluripotent stem cells, involve invasive procedures or complex differentiation protocols, respectively. Here, we show that a set of four transcription factors, Hnf4α, Foxa3, Gata6, and Cdx2, can directly reprogram mouse fibroblasts to acquire the identity of fetal intestine-derived progenitor cells (FIPCs). These induced FIPCs (iFIPCs) form spherical organoids that develop into adult-type budding organoids containing cells with intestinal stem cell properties. The resulting stem cells produce all intestinal epithelial cell lineages and undergo self-renewing cell divisions. After transplantation, the induced spherical and budding organoids can reconstitute colonic and intestinal epithelia, respectively. The same combination of four defined transcription factors can also induce human iFIPCs. This alternative approach for producing intestinal organoids may well facilitate application for disease analysis and therapy development.

Myofibroblasts Derived from Hepatic Progenitor Cells Create the Tumor Microenvironment.

Hepatic progenitor cells (HPCs) appear in response to several types of chronic injury in the human and rodent liver that often develop into liver fibrosis, cirrhosis, and primary liver cancers. However, the contribution of HPCs to the pathogenesis and progression of such liver diseases remains controversial. HPCs are generally defined as cells that can differentiate into hepatocytes and cholangiocytes. In this study, however, we found that HPCs isolated from the chronically injured liver can also give rise to myofibroblasts as a third type of descendant. While myofibroblast differentiation from HPCs is not significant in culture, during tumor development, HPCs can contribute to the formation of the tumor microenvironment by producing abundant myofibroblasts that might form a niche for tumor growth and survival. Thus, HPCs can be redefined as cells with a potential for differentiation into myofibroblasts that is specifically activated during tumor formation.

Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease.

Acquisition of lipid metabolic capability in hepatocyte-like cells directly induced from mouse fibroblasts.

Recently, the numbers of patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have increased worldwide. NAFLD and NASH are known as risk factors for liver cirrhosis and hepatocellular carcinoma. Because many factors can promote the progression of NAFLD and NASH, the treatment of these patients involves various strategies. Thus, it is desired that drugs for patients with NAFLD and NASH should be developed more easily and rapidly using cultures of primary hepatocytes. However, it is difficult to use hepatocytes as a tool for drug screening, because these cells cannot be functionally maintained in culture. Thus, in this study, we sought to examine whether induced hepatocyte-like (iHep) cells, which were directly induced from mouse dermal fibroblasts by infection with a retrovirus expressing Hnf4α and Foxa3, possess the potential for lipid metabolism, similar to hepatocytes. Our data showed that iHep cells were capable of synthesizing lipids from a cis-unsaturated fatty acid, a trans-unsaturated fatty acid, and a saturated fatty acid, accumulating the synthesized lipids in cellular vesicles, and secreting the lipids into the culture medium. Moreover, the lipid synthesis in iHep cells was significantly inhibited in cultures with lipid metabolism improvers. These results demonstrate that iHep cells could be useful not only for screening of drugs for patients with NAFLD and NASH, but also for elucidation of the mechanisms underlying hereditary lipid metabolism disorders, as an alternative to hepatocytes.