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.

Direct Conversion of Human Endothelial Cells Into Liver Cancer-Forming Cells Using Nonintegrative Episomal Vectors.

Liver cancer is an aggressive cancer associated with a poor prognosis. Development of therapeutic strategies for liver cancer requires fundamental research using suitable experimental models. Recent progress in direct reprogramming technology has enabled the generation of many types of cells that are difficult to obtain and provide a cellular resource in experimental models of human diseases. In this study, we aimed to establish a simple one-step method for inducing cells that can form malignant human liver tumors directly from healthy endothelial cells using nonintegrating episomal vectors. To screen for factors capable of inducing liver cancer-forming cells (LCCs), we selected nine genes and one short hairpin RNA that suppresses tumor protein p53 (TP53) expression and introduced them into human umbilical vein endothelial cells (HUVECs), using episomal vectors. To identify the essential factors, we examined the effect of changing the amounts and withdrawing individual factors. We then analyzed the proliferation, gene and protein expression, morphologic and chromosomal abnormality, transcriptome, and tumor formation ability of the induced cells. We found that a set of six factors, forkhead box A3 (FOXA3), hepatocyte nuclear factor homeobox 1A (HNF1A), HNF1B, lin-28 homolog B (LIN28B), MYCL proto-oncogene, bHLH transcription factor (L-MYC), and Kruppel-like factor 5 (KLF5), induced direct conversion of HUVECs into LCCs. The gene expression profile of these induced LCCs (iLCCs) was similar to that of human liver cancer cells, and these cells effectively formed tumors that resembled human combined hepatocellular-cholangiocarcinoma following transplantation into immunodeficient mice. Conclusion: We succeeded in the direct induction of iLCCs from HUVECs by using nonintegrating episomal vectors. iLCCs generated from patients with cancer and healthy volunteers will be useful for further advancements in cancer research and for developing methods for the diagnosis, treatment, and prognosis of liver cancer.

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.

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.

Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver.

The proliferation of biliary lineage cells in chronic liver diseases, which leads to formation of primitive ductules in portal areas of the hepatic lobule, may be important not only for liver regeneration, but also for initiation of liver cancer. Thus, understanding how these primitive ductular cells emerge and proliferate in chronically injured liver holds promise for development of therapeutic strategies for liver diseases. However, the origin of these primitive ductular cells remains controversial. Here, we use a method for genetic lineage tracing to determine the origin of cells that form primitive ductules in a mouse model of chronic liver injury. Our results show that hepatocytes, rather than cholangiocytes, are the major source of cells for the primitive ductules formed in response to chronic liver damage. Moreover, activation of the Notch-Hes1 signaling axis is important for conversion of hepatocytes into primitive ductular cells in chronically injured liver. These findings should be valuable in elucidating the mechanism of liver regeneration associated with the fate-conversion of hepatocytes and in developing therapeutic strategies for liver diseases.

Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary malignancy in the liver. ICC has been classified as a malignant tumor arising from cholangiocytes; however, the co-occurrence of ICC and viral hepatitis suggests that ICC originates in hepatocytes. In order to determine the cellular origin of ICC, we used a mouse model of ICC in which hepatocytes and cholangiocytes were labeled with heritable, cell type­specific reporters. Our studies reveal that ICC is generated by biliary lineage cells derived from hepatocytes, rather than cholangiocytes. Additionally, we found that Notch activation is critical for hepatocyte conversion into biliary lineage cells during the onset of ICC and its subsequent malignancy and progression. These findings will help to elucidate the pathogenic mechanism of ICC and to develop therapeutic strategies for this refractory disease.

EGF signaling activates proliferation and blocks apoptosis of mouse and human intestinal stem/progenitor cells in long-term monolayer cell culture.

The homeostatic renewal of the intestinal epithelium depends on regulation of proliferation and differentiation of stem/progenitor cells residing in a specific site, called the 'stem cell niche.' Thus, the reconstitution of the microenvironment of the stem cell niche may allow us to maintain intestinal stem/progenitor cells in culture for a longer period. Although epidermal growth factor (EGF) is conventionally used as a supplement of intestinal epithelial cell culture, little has been known regarding a role of EGF signaling in a stem/progenitor cell population. In this study, we attempted to clarify the role of EGF signaling in intestinal stem/progenitor cells, and to establish a culture system in which these cells could be maintained with normal differentiation potential. We first examined the expression pattern of EGF and its receptor, EGFR, and inhibited EGF signaling in mouse intestines. Next, we cultured intestinal cells isolated from mouse and human intestines in the presence of EGF and analyzed the function of EGF signaling in cultured cells. In both embryonic and adult mouse intestines, EGFR and EGF were expressed in immature epithelial cells and adjacent fibroblasts, respectively, and EGF signaling was essential to activate proliferation and inhibit apoptosis of intestinal stem/progenitor cells. Activation of EGF signaling also stimulated proliferation and suppressed apoptosis, both of which are necessary to maintain mouse and human intestinal epithelial cells in culture. Moreover, in these cultured epithelial cells, putative intestinal stem/progenitor cells persisted longer, and gave rise to different types of differentiated intestinal epithelial cells. We conclude that EGF signaling is indispensable for activation of proliferation and inhibition of unexpected cell death, not only in the intestinal stem cell niche, but also in culture of primitive intestinal epithelial cells.

Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.

UNLABELLED: The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self-renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell-based analysis to examine precisely bilineage differentiation potential and self-renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133(+) cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo. Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self-renewing cell divisions. The clonogenic progeny of an LC-forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC-forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell-like capacity of isolated and clonally cultured CD133(+) LC-forming cells. CONCLUSION: Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis.

Tbx3 controls the fate of hepatic progenitor cells in liver development by suppressing p19ARF expression.

Although the T-box family of transcription factors function in many different tissues, their role in liver development is unknown. Here we show that Tbx3, the T-box gene that is mutated in human ulnar-mammary syndrome, is specifically expressed in multipotent hepatic progenitor cells, ;hepatoblasts', isolated from the developing mouse liver. Tbx3-deficient hepatoblasts presented severe defects in proliferation as well as uncontrollable hepatobiliary lineage segregation, including the promotion of cholangiocyte (biliary epithelial cell) differentiation, which thereby caused abnormal liver development. Deletion of Tbx3 resulted in the increased expression of the tumor suppressor p19(ARF) (Cdkn2a), which in turn induced a growth arrest in hepatoblasts and activated a program of cholangiocyte differentiation. Thus, Tbx3 plays a crucial role in controlling hepatoblast proliferation and cell-fate determination by suppressing p19(ARF) expression and thereby promoting liver organogenesis.

Cyclization of aryl acyl radicals generated from S-(4-cyano)phenyl thiolesters by a nickel complex catalyzed electroreduction.

Aromatic acyl radicals generated from S-(4-cyano)phenyl 2-alkenylthiobenzoate by a nickel complex catalyzed electroreduction undergo 5- and 6-exo cyclization to give 1-indanone and dihydro-1-naphthalenone derivatives, respectively.