Transdifferentiation Of Hepatocytes Research Articles

Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.

Hepatocyte-to-cholangiocyte conversion occurs through transdifferentiation independently of proliferation in zebrafish.

Injury to biliary epithelial cells (BECs) lining the hepatic bile ducts leads to cholestatic liver diseases. Upon severe biliary damage, hepatocytes can convert to BECs, thereby contributing to liver recovery. Given a potential of augmenting this hepatocyte-to-BEC conversion as a therapeutic option for cholestatic liver diseases, it will be important to thoroughly understand the cellular and molecular mechanisms of the conversion process. Towards this aim, we have established a zebrafish model for hepatocyte-to-BEC conversion by employing Tg(fabp10a:CFP-NTR) zebrafish with a temporal inhibition of Notch signaling during regeneration. Cre/loxP-mediated permanent and H2B-mCherry-mediated short-term lineage tracing revealed that in the model, all BECs originate from hepatocytes. During the conversion, BEC markers are sequentially induced in the order of Sox9b, Yap/Taz, Notch activity/ epcam , and Alcama/ krt18 ; the expression of the hepatocyte marker Bhmt disappears between the Sox9b and Yap/Taz induction. Importantly, live time-lapse imaging unambiguously revealed transdifferentiation of hepatocytes into BECs: hepatocytes convert to BECs without transitioning through a proliferative intermediate state. In addition, using compounds and transgenic and mutant lines that modulate Notch and Yap signaling, we found that both Notch and Yap signaling are required for the conversion even in Notch- and Yap-overactivating settings. Hepatocyte-to-BEC conversion occurs through transdifferentiation independently of proliferation, and Notch and Yap signaling control the process in parallel with a mutually positive interaction. The new zebrafish model will further contribute to a thorough understanding of the mechanisms of the conversion process.

ZO-2/Tjp2 suppresses Yap and Wwtr1/Taz-mediated hepatocyte to cholangiocyte transdifferentiation in the mouse liver

TJP2/ZO-2-inactivating mutations in humans cause progressive cholestatic liver disease. Liver-specific deletion of Tjp2 in the mouse (Tjp2 cKO mice) leads to mild progressive cholestasis without an overt degradation of the bile-blood barrier (BBB). These mice are more susceptible to cholic acid (CA) induced liver injury. Interestingly, while initially also more susceptible, Tjp2 cKO mice develop tolerance to a DDC-supplemented diet. The DDC diet induces an exacerbated ductular reaction in Tjp2 cKO mice, which arises from the transdifferentiation of hepatocytes to cholangiocytes. Consequently, this transdifferentiation is only observed if Tjp2 is inactivated in hepatocytes, but not if deleted in cholangiocytes. The DDC-diet-induced hepatocyte transdifferentiation in Tjp2 cKO mice requires Yap and Wwtr1/Taz, whose protein expression is upregulated in hepatocytes lacking Tjp2, but is independent of Notch2. Although inactivating Tjp2 is sufficient for the upregulation of Yap and Wwtr1/Taz protein, efficient transdifferentiation requires the DDC-diet insult. Thus, Tjp2 negatively regulates Yap/Taz-mediated transdifferentiation of hepatocytes to cholangiocytes in response to DDC-diet-induced liver injury. Furthermore, transdifferentiation is regulated at multiple levels and the type of injury inflicted on the Tjp2 deficient liver plays an important role in the resulting pathophysiology.

Features of Ductular Reaction in Rats with Extrahepatic Cholestasis.

Ductular reaction develops during liver regeneration, fibrosis, and carcinogenesis. However, the types, stages of formation, and topography of ductular profiles in various pathologies remain insufficiently studied. Using the model of common bile duct occlusion, we showed that the number and topography of ductular profiles are closely related to the duration of biliary obstruction. The ductular profiles can be located inside the portal tract, along the existing bile ducts, and/or intramurally, around the portal vein, periportally, inside the lobules, in the portocaval fibrous connections, in the adventitia of the hepatic veins, in the septs connecting the portal tracts, and also in the "portal plate" of the liver. The ductular profiles can be formed as a result of expansion of existing bile ducts, cholangiocyte proliferation, as well as transdifferentiation of hepatocytes and activation of mesenchymal stem cells.

TNF receptor-related factor 3 inactivation promotes the development of intrahepatic cholangiocarcinoma through NF-κB-inducing kinase-mediated hepatocyte transdifferentiation.

Intrahepatic cholangiocarcinoma (ICC) is a deadly but poorly understood disease, and its treatment options are very limited. The aim of this study was to identify the molecular drivers of ICC and search for therapeutic targets. We performed a Sleeping Beauty transposon-based in vivo insertional mutagenesis screen in liver-specific Pten -deficient mice and identified TNF receptor-related factor 3 ( Traf3 ) as the most significantly mutated gene in murine ICCs in a loss-of-function manner. Liver-specific Traf3 deletion caused marked cholangiocyte overgrowth and spontaneous development of ICC in Pten knockout and KrasG12D mutant mice. Hepatocyte-specific, but not cholangiocyte-specific, Traf3 -deficient and Pten -deficient mice recapitulated these phenotypes. Lineage tracing and single-cell RNA sequencing suggested that these ICCs were derived from hepatocytes through transdifferentiation. TRAF3 and PTEN inhibition induced a transdifferentiation-like phenotype of hepatocyte-lineage cells into proliferative cholangiocytes through NF-κB-inducing kinase (NIK) up-regulation in vitro. Intrahepatic NIK levels were elevated in liver-specific Traf3 -deficient and Pten -deficient mice, and NIK inhibition alleviated cholangiocyte overgrowth. In human ICCs, we identified an inverse correlation between TRAF3 and NIK expression, with low TRAF3 or high NIK expression associated with poor prognosis. Finally, we showed that NIK inhibition by a small molecule inhibitor or gene silencing suppressed the growth of multiple human ICC cells in vitro and ICC xenografts in vivo. TRAF3 inactivation promotes ICC development through NIK-mediated hepatocyte transdifferentiation. The oncogenic TRAF3-NIK axis may be a potential therapeutic target for ICC.

YAP1 activation and Hippo pathway signaling in the pathogenesis and treatment of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, is a highly lethal epithelial cell malignancy exhibiting features of cholangiocyte differentiation. iCCAs can potentially develop from multiple cell types of origin within liver, including immature or mature cholangiocytes, hepatic stem cells/progenitor cells, and from transdifferentiation of hepatocytes. Understanding the molecular mechanisms and genetic drivers that diversely drive specific cell lineage pathways leading to iCCA has important biological and clinical implications. In this context, activation of the YAP1-TEAD dependent transcription, driven by Hippo-dependent or -independent diverse mechanisms that lead to the stabilization of YAP1 is crucially important to biliary fate commitment in hepatobiliary cancer. In preclinical models, YAP1 activation in hepatocytes or cholangiocytes is sufficient to drive their malignant transformation into iCCA. Moreover, nuclear YAP1/TAZ is highly prevalent in human iCCA irrespective of the varied etiology, and significantly correlates with poor prognosis in iCCA patients. Based on the ubiquitous expression and diverse physiologic roles for YAP1/TAZ in the liver, recent studies have further revealed distinct functions of active YAP1/TAZ in regulating tumor metabolism, as well as the tumor immune microenvironment. In the current review, we discuss our current understanding of the various roles of the Hippo-YAP1 signaling in iCCA pathogenesis, with a specific focus on the roles played by the Hippo-YAP1 pathway in modulating biliary commitment and oncogenicity, iCCA metabolism, and immune microenvironment. We also discuss the therapeutic potential of targeting the YAP1/TAZ-TEAD transcriptional machinery in iCCA, its current limitations, and what future studies are needed to facilitate clinical translation.

Abstract 2927: Inactivation of traf3 promotes intrahepatic cholangiocarcinoma development via hepatocyte trans-differentiation

Abstract Aim: Intrahepatic cholangiocarcinoma (ICC) is a deadly cancer but little is known about its carcinogenesis mechanism. Insertional mutagenesis screen using sleeping beauty transposon (SBT) is a versatile tool to identify cancer genes in a high-throughput manner. We aimed to discover genes driving ICC by SBT mutagenesis screen in mice. Methods: The mice with hundreds copies of movable mutagenic SBT were crossed with the mice knocking out Pten, a recurrently mutated cancer gene in human ICC, in hepatocytes and intrahepatic cholangiocytes using Albumin-Cre (Alb-Cre). They were aged and all the SBT-induced liver tumors were histologically assessed. Among the tumors with CCC component, frequently mutated genes were identified by the sequencing of transposon insertion sites. We then generated the mice with deletion of Traf3 and Pten in the liver or adult hepatocytes or intrahepatic cholangiocytes using Albumin-Cre (L-DKO) or Alb-CreERT2 (H-DKO) or K19-CreERT2 (C-DKO) mice, respectively. Adult hepatocytes-specific Traf3/Pten DKO mice was also generated by hydrodynamic tail vein injection (HTVi-DKO) or adeno-associated virus (AAV-DKO)-mediated Cre recombinase delivery. To identify cell origin of ICC, lineage tracing was performed by crossing H-DKO mice with ROSA26-LacZ reporter mice(H-DKO/LacZ). Single cell RNA-sequencing (scRNA-seq) was performed to clarify differentiation process of liver cells in the L-DKO mice. Results: Transposon mutagenesis significantly accelerated liver tumorigenesis in the Pten-deficient mice. Among 20 tumors rich in CCC component, Traf3 was most frequently mutated by SBT in a loss-of-function manner. L-DKO mice showed marked biliary hyperplasia at 4 weeks of age and developed ICC at 3 months of age. While H-DKO, HTVi-DKO and AAV-DKO mice also phenocopied L-DKO mice, C-DKO mice did not recapitulate those phenotypes. Proliferating bile ducts and cholangiocarcinoma cells in H-DKO/LacZ mice were positive for β-galactosidase staining, indicating that they were originated from hepatocytes. ScRNA-seq identified unique cell population with both hepatocytes and cholangiocyte properties only in the liver of L-DKO mouse but not in the wild-type mouse. Trajectory analysis suggested that they were transdifferentiated from mature hepatocytes. In vitro experiments showed that Traf3/Pten inhibition significantly upregulated cholangiocyte markers in murine organoid-derived and human immortalized hepatocytes. Both L-DKO and H-DKO mice showed elevation of TNFα in the liver and its deletion significantly ameliorated development of ICC in the L-DKO mice. Human ICC patients with low intra-tumor Traf3 levels had more advanced stages and poorer prognosis than those with its high levels. Conclusion: Inactivation of Traf3 promotes development of ICC via hepatocyte trans-differentiation in mice and may contribute to ICC pathogenesis in humans. Citation Format: Yuto Shiode, Takahiro Kodama, Hayato Hikita, Ryotaro Sakamori, Tomohide Tatsumi, Tetsuo Takehara. Inactivation of traf3 promotes intrahepatic cholangiocarcinoma development via hepatocyte trans-differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2927.

Loss of hepatocyte identity following aberrant YAP activation: A key mechanism in alcoholic hepatitis

Alcoholic hepatitis (AH) is a life-threatening disease with limited therapeutic options, as the molecular mechanisms leading to death are not well understood. This study evaluates the Hippo/Yes-associated protein (YAP) pathway which has been shown to play a role in liver regeneration. The Hippo/YAP pathway was dissected in explants of patients transplanted for AH or alcohol-related cirrhosis and in control livers, using RNA-seq, real-time PCR, western blot, immunohistochemistry and transcriptome analysis after laser microdissection. We transfected primary human hepatocytes with constitutively active YAP (YAPS127A) and treated HepaRG cells and primary hepatocytes isolated from AH livers with a YAP inhibitor. We also used mouse models of ethanol exposure (Lieber de Carli) and liver regeneration (carbon tetrachloride) after hepatocyte transduction of YAPS127A. In AH samples, RNA-seq analysis and immunohistochemistry of total liver and microdissected hepatocytes revealed marked downregulation of the Hippo pathway, demonstrated by lower levels of active MST1 kinase and abnormal activation of YAP in hepatocytes. Overactivation of YAP in hepatocytes invitro and invivo led to biliary differentiation and loss of key biological functions such as regeneration capacity. Conversely, a YAP inhibitor restored the mature hepatocyte phenotype in abnormal hepatocytes taken from patients with AH. In ethanol-fed mice, YAP activation using YAPS127A resulted in a loss of hepatocyte differentiation. Hepatocyte proliferation was hampered by YAPS127A after carbon tetrachloride intoxication. Aberrant activation of YAP plays an important role in hepatocyte transdifferentiation in AH, through a loss of hepatocyte identity and impaired regeneration. Thus, targeting YAP is a promising strategy for the treatment of patients with AH. Alcoholic hepatitis is characterized by inflammation and a life-threatening alteration of liver regeneration, although the mechanisms behind this have not been identified. Herein, we show that liver samples from patients with alcoholic hepatitis are characterized by profound deregulation of the Hippo/YAP pathway with uncontrolled activation of YAP in hepatocytes. We used human cell and mouse models to show that inhibition of YAP reverts this hepatocyte defect and could be a novel therapeutic strategy for alcoholic hepatitis.

P‐Ezrin (Thr567) synergizes with Akt signaling to accelerate Yap mediated cholangiocarcinogenesis, but correlates with favorable prognosis in human intrahepatic cholangiocarcinoma(ICC) patients.

Ezrin phosphorylation is directly involved in regulation of Hippo pathway, Yap levels and growth rates of normal and neoplastic hepatocytes. It also has been shown that coordinated activation of Akt and Yap induces intrahepatic cholangiocarcinoma (ICC) in mice. In this study, we aimed to illustrate if surrogate maker of Yap, p-Ezrin(Thr567) could work with Akt to establish cholangiocarcinogenesis, and its potential translational significance. Initially activated Akt hydrodynamic tail vein injection mouse model was established and studied. Following that, we generated two mouse models by the same approach with mutant Ezrin (T567D) (which mimics p-Ezrin(Thr567)) or wild type Ezrin, in association with or without activated form of Akt. After 3 months, while forced co-expression of wild type Ezrin and Akt in the C57 mouse strain did not induce any appreciable abnormality, mutant Ezrin/Akt mouse model was associated with development of macroscopically visible tumors in the liver. After 5 months, wild type Ezrin and Akt in the C57 mouse strain induced macroscopically visible tumors in the liver. Surprisingly, over expression activated Akt alone in the FVB mouse strain induced macroscopically visible tumor only after 21 weeks of injection. An activated Akt associated “balloon cell” like hepatocytes phenomenon was observed and oil red staining was positive. By H&E staining, these tumor cells in the C57 mouse strain had the histology of purely cholangiocyte neoplasms. However, the countparts in the FVB mouse showed a mixed cholangiocyte and hepatocyte derivation, mixed tumor types, including hepatic adenoma, hepatocellular carcinoma and cholangiocarcinoma, and mixed stages of variant neoplasms. In addition, an intense infiltrating immune cell response along the edge of the neoplasms was observed specifically in the mutant Ezrin/Akt FVB mouse model at all the time points (3 month, 4 month, and 5 month) with a positive Ezrin staining. All those malignant tumor cells were consistently positive for mutant Ezrin (T567D)(GFP), and Akt (HA tag) positive. We also observed Yap, CK19 and PCNA positive staining specifically on these tumor cells. ICC tissue microarray revealed only 24% patients were p-Ezrin(Thr567) positive, this positivity correlated with lower TNM stage, lower tumor grade, and higher Yap expression. In addition, cBioPortal database showed there were total 5.3% ICC patients tested FAT1 and NF2 genomic abnormalities, and 10% ICC patients tested PIK3CA and PTEN genomic abnormalities. Conclusions We successfully established a purely ICC mouse model and a mixed HCC/ICC mouse model, which could be clearly identified macroscopically and microscopically. The fact that these neoplasms arose after hydrodynamic injection, strongly suggests that the cholangiocarcinomas observed arose from hepatocyte to cholangiocyte transdifferentiation. The finding has mechanistic and potentially therapeutic applications in understanding and regulating and trans-differentiation of hepatocytes, growth of cholangiocytes and Intrahepatic Cholangiocarcinoma (ICC) pathogenesis.

An Efficient and Footprint-Free Protocol for the Transdifferentiation of Hepatocytes Into Insulin-Producing Cells With IVT mRNAs.

BackgroundDirect transdifferentiation of adult somatic cells into insulin-producing cells (IPCs) is a promising approach for cell-based therapies for type 1 diabetes mellitus. Liver cells are an ideal source for generating IPCs because they have regenerative ability and a developmental process similar to that of the pancreas. Pancreas versus liver fate is regulated by TALE homeoprotein (TGIF2) during development. Here, we wanted to investigate whether TGIF2 could enhance the efficiency of transdifferentiation of hepatocytes into IPCs induced by three pancreatic transcription factors (pTFs), i.e., Pdx1, NeuroD, and Mafa, which are crucial for pancreatic development in the embryo.MethodsThe in vitro transcribed (IVT) mRNAs of TGIF2 and the three pTFs were synthesized in vitro and sequentially supplemented in hepatocytes. On day 6, the expression of transcription factors was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and insulin expression was detected by immunofluorescence. Glucose-stimulated insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). The key genes controlling cell polarity and the Wnt/PCP signaling pathway were assayed by qRT-PCR, and the level of JNK protein phosphorylation, which regulates the Wnt/PCP signaling pathway, was detected by western blotting.ResultsIVT mRNAs could be efficiently transfected into hepatocytes. Quantitative real-time polymerase chain reaction results revealed that compared with ectopic expression of the three pTFs alone, ectopic expression of the three pTFs plus TGIF2 could strongly reduce hepatic gene expression and subsequently improve the induction of a set of pancreatic genes. Immunofluorescence analysis showed that TGIF2 expression could double the transdifferentiation yield; 30% of the cells were insulin positive if induced by TGIF2 plus the 3 pTFs, while only 15% of the cells were insulin positive if induced by the three pTFs alone. ELISA analysis confirmed that glucose-stimulated insulin secretion was less efficient after transfection with the three pTFs alone. The differentiated cells derived from the addition of TGIF2 mRNA could form islet-like clusters. By contrast, the cells differentiated with the three pTFs did not form clusters under the same conditions. Tgif2 induced transdifferentiation more efficiently by remodeling the expression of genes in the Wnt/PCP pathway. Overexpression of TGIF2 in hepatocytes could activate the expression of key genes controlling cell polarity and genes in the Wnt/PCP signaling pathway, increasing the level of JNK protein phosphorylation.ConclusionsOur study established a novel footprint-free protocol for efficient transdifferentiation of hepatocytes into IPCs using IVT mRNAs of TGIF2 and 3 pTFs, which paved the way toward a clinical application.

Determining the effects of Wnt signaling in the alleviation of cholestasis via the promotion of hepatocyte transdifferentiation

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease characterized by bile duct inflammation and fibrosis, resulting in end‐stage liver disease and reduced life expectancy. Therefore, an effective treatment for PSC is needed. Hepatocytes exhibit remarkable plasticity and are known to be capable of transdifferentiating into cholangiocytes in models of biliary injury, which could create de novo ducts, repair damaged cholangiocytes, or contribute to bile detoxification. Previous studies utilizing in vitro organoid cultures and genetic mouse models found that β‐catenin and downstream targets are upregulated in hepatic organoid cultures, and mice expressing excess β‐catenin in the liver had an increased number of hepatocytes expressing cholangiocyte markers compared to wild type (WT) when subjected to cholestatic injury. These findings led to the hypothesis that Wnt/ß‐catenin signaling drives hepatocyte‐to‐cholangiocyte transdifferentiation during biliary injury. To test this hypothesis, we utilized lineage tracing in hepatic organoid cultures and TG mice expressing a mutated non‐degradable form of β‐catenin (S45D) in liver. We show that hepatocytes transdifferentiate to cholangiocytes in organoid cultures, and the cholangiocytes present are not from native contaminating cholangiocytes. We determined that TG mice fed DDC diet, which induces bile stasis, have: 1) improved serum ALP over time, indicating less biliary injury; 2) increased bile output compared to WT mice fed DDC diet; and 3) bile ducts populated with hepatocyte‐derived cholangiocytes. Through these studies we demonstrate that Wnt/ß‐catenin signaling catalyzes hepatocyte‐to‐cholangiocyte transdifferentiation, and activation of this pathway alleviates cholestasis in mouse models of PSC.Support or Funding InformationNIH grant 1R01DK103775NIH training grant T32EB0010216NIH grant 1F31DK118802‐01

Nuclear Phosphoinositide 3‐kinase delta (PIK3CD) Increases with Conditional Deletion of Hepatocellular Integrin Linked Kinase

IntroductionThe extracellular matrix (ECM) is important for survival, differentiation, and normal functioning of cells within the liver; integrins are key signaling receptors in this process. Previous data from our lab has shown that with hepatocyte specific knockout of integrin linked kinase (ILK KO), there is hepatocyte proliferation, increased matrix deposition, unorganized biliary cell/ductal proliferation, and possible transdifferentiation of hepatocytes to cholangiocytes; this led us to investigate the signaling pathways downstream of hILK KO that might be responsible for the observed phenotype, and we uncovered a potential central role for Phosphoinositide 3‐kinase (PI3K) delta (PIK3CD), a variant of PI3K that is considered to be leukocyte specific.MethodsMice with germ‐line loss of hepatocellular ILK were generated by crossing double floxed ILK mice (ILKfl/fl) with mice expressing cre recombinase under an afp enhancer‐albumin promoter (Cre+/−) to obtain ILKfl/fl:Cre+/− (ILK KO) and ILKfl/fl:Cre−/− (WT). Data array analyses were performed on livers from 7‐ and 14‐week old chronic ILK KO and WT mice. RNAs of interest were identified and then further examined by western blot analyses, immunohistochemical staining, and induction of acute loss of ILK using AAV8 cre in ILKfl/fl mice.ResultsExamination of the 7‐week RNA array analysis suggested a central role for PIK3CD in the observed hILK phenotype. Unexpectedly, using immunohistochemistry we detected prominent staining of PIK3CD protein in ILK KO hepatocytes. The presence of PIK3CD was verified in cultures of wild type hepatocytes where inhibition of PIK3CD resulted in a decrease in both pAKT (a downstream target) and Cyclin D1. Western blot analyses of whole livers also uncovered a size variant that is only observed in isolated nuclei. Additionally, at 7 days after the acute removal of ILK, we can observe staining for the cholangiocyte specific marker, CK19, in hepatocytes as well as strong PIK3CD staining in regions with spontaneous duct formation.ConclusionOur RNA and protein data from ILK KO mice have 1) revealed PIK3CD, a leukocyte specific protein, to be hepatocellular 2) shown that a size variant of PIK3CD exists in the nuclei of hepatocytes and 3) uncovered a potential relationship between PIK3CD in hepatocytes and their transdifferentiation to biliary cells. Currently, we are using an in vitro model of trans‐differentiation in an attempt to further explore these preliminary findings.Support or Funding InformationCATER NIH T32 EB001026.

Novel Model of Bile Duct Paucity Demonstrates the Critical Role of Yap1 in Biliary Morphogenesis in Development and Regeneration

BackgroundYes‐associated protein 1 (Yap1) is a critical regulator of liver embryonic development, regeneration and tumorigenesis. Previous studies have shown that Yap1 regulates bile duct formation and hepatocyte transdifferentiation to cholangiocytes, but the mechanisms by which Yap1 regulates biliary morphogenesis as well as the functions of Yap1 in early embryonic liver development remain unknown.MethodsTo elucidate the role of Yap1 in early liver development, we developed a novel mouse model (Yap1 KO) in which Yap1 is deleted from the foregut endoderm progenitors by E12.5 using the Foxa3 promoter to drive Cre recombinase expression. Here, we comprehensively characterize the Yap1 KO mice through histology, whole organ tissue clearing and confocal microscopy and additional functional techniques.ResultsYap1 KO mice lack Yap1 in the liver from early development. Strong nuclear expression of Yap1 that was seen in hepatoblasts in WT livers at E14.5 was clearly absent in the KO mice. Although Yap1 KO mice were viable and have fully formed liver and digestive organs, they exhibited jaundice and stunted growth. Histological analysis identified a failure of intrahepatic bile duct formation. Yap1 deletion did not block Notch activation as shown by the appearance of Hes1‐positive and Sox9‐positive ductal plate cells in both WT and KO mice. Rather, Yap1 deletion prevented the morphological assembly of a two‐cell layer with a lumen from an asymmetrical single‐layered ductal plate. Surprisingly, KO mice survive long‐term with normalization of serum liver enzymes despite persistent cholestatic injury, suggesting hepatocyte adaptation to injury. While a recent study of Notch‐deficient livers (a model of Alagille syndrome) described complete de novo hepatocyte‐derived generation of bile ducts by 4 months of age, Yap1‐deficient livers fail to regenerate bile ducts by 8 months of age. Liver tissue clearing and 3D whole liver immunofluorescence imaging showed severely abbreviated biliary tree in KO mice relative to WT. Notably, we observe the appearance of a ductular reaction in the hilar region of the liver consisting of Yap1‐positive ducts surrounded by fibrosis and inflammation. Although all hepatocytes remain Yap1‐negative, the extrahepatic biliary tree remains unaffected by the Cre and is Yap1‐positive, suggesting a potential source of Yap1‐positive ductular cells.ConclusionsOur results demonstrate that Yap1 is dispensable for early development of the liver and foregut but is critical for biliary morphogenesis. We further show absence of hepatocyte‐to‐cholangiocyte transdifferentiation in Yap1 KO. Thus, Yap1 is essential for biliary morphogenesis regardless of the cellular origin, whether from hepatoblasts in development or from hepatocytes in liver regeneration.Support or Funding InformationThis work was supported by NIH grants 1R01DK62277, 1R01DK100287, 1R01DK116993 and Endowed Chair for Experimental Pathology to S.P.M, 1P30DK120531‐01 to the Pittsburgh Liver Research Center, and T32EB0010216 to LM.

Inherited Cholestatic Diseases in the Era of Personalized Medicine.

http://aasldpubs.onlinelibrary.wiley.com/hub/journal/10.1002/(ISSN)2046-2484/video/15-3-reading-mack a video presentation of this article.

IL‐8 induces transdifferentiation of mature hepatocytes toward the cholangiocyte phenotype

The adult mammalian liver exhibits a remarkable regenerative capacity, with different modes of regeneration according to the type and extent of injury. Hepatocyte–cholangiocyte biphenotypic liver progenitor cell populations appear under conditions of excessive injury. It has been reported that mature hepatocytes can transdifferentiate toward a cholangiocyte phenotype and be a cellular source of progenitor cell populations. Here, we determined that among various plasma cytokines, interleukin (IL)‐8 levels were significantly elevated in acute liver failure and severe acute liver injury patients. In vitro assays revealed that administration of IL‐8 homologues increases the expression of Sry HMG box protein 9 (SOX9). In liver biopsies of acute liver injury patients, we observed the appearance of SOX9‐positive biphenotypic hepatocytes accompanied by elevation of plasma IL‐8 levels. Our results suggest that IL‐8 regulates the phenotypic conversion of mature hepatocytes toward a cholangiocyte phenotype.

Mir-101-3p Downregulation Promotes Fibrogenesis by Facilitating Hepatic Stellate Cell Transdifferentiation During Insulin Resistance.

Insulin resistance (IR) and microRNAs (miRNAs), which regulate cell-to-cell communication between hepatocytes and hepatic stellate cells (HSCs), may intertwine in nonalcoholic fatty liver disease (NAFLD) pathogenesis. The aim of this study was to evaluate whether epigenetics and environmental factors interact to promote progressive NAFLD during IR. We examined the miRNA signature in insulin receptor haploinsufficient (InsR+/−) and wild-type (wt) HSCs by RNAseq (n = 4 per group). Then, we evaluated their impact in an IR-NASH (nonalcoholic steatohepatitis) model (InsR+/− mice fed standard or methionine choline deficient (MCD) diet, n = 10 per group) and in vitro. InsR+/− HSCs displayed 36 differentially expressed miRNAs (p < 0.05 vs. wt), whose expression was then analyzed in the liver of InsR+/− mice fed an MCD diet. We found that miR-101-3p negatively associated with both InsR+/− genotype and MCD (p < 0.05) and the histological spectrum of liver damage (p < 0.01). miR-101-3p was reduced in InsR+/− hepatocytes and HSCs and even more in InsR+/− cells exposed to insulin (0.33 µM) and fatty acids (0.25 mM), resembling the IR-NASH model. Conversely, insulin induced miR-101-3p expression in wt cells but not in InsR+/− ones (p < 0.05). In conclusion, IR combined with diet-induced liver injury favors miR-101-3p downregulation, which may promote progressive NAFLD through HSC and hepatocyte transdifferentiation and proliferation.

YAP Activation Drives Liver Regeneration after Cholestatic Damage Induced by Rbpj Deletion.

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.

Chimeric liver transplantation reveals interspecific graft remodelling

A major limitation in the field of liver transplantation is the shortage of transplantable organs. Chimeric animals carrying human tissue have the potential to solve this problem. However, currently available chimeric organs retain a high level of xenogeneic cells, and the transplantation of impure organs needs to be tested. We created chimeric livers by injecting Lewis rat hepatocytes into C57Bl/6Fah-/-Rag2-/-Il2rg-/- mice, and further transplanted them into newly weaned Lewis rats (45 ± 3 g) with or without suboptimal immunosuppression (tacrolimus 0.6 mg/kg/day for 56 or 112 days). Control donors included wild-type C57Bl/6 mice (xenogeneic) and Lewis rats (syngeneic). Without immunosuppression, recipients of chimeric livers experienced acute rejection, and died within 8 to 11 days. With immunosuppression, they all survived for >112 days with normal weight gain compared to syngeneic controls, while all xenogeneic controls died within 98 days due to rejection with Banff scores >6 (p = 0.0014). The chimeric grafts underwent post-transplant remodelling, growing by 670% on average. Rat hepatocytes fully replaced mouse hepatocytes starting from day 56 (absence of detectable mouse serum albumin, histological clearance of mouse hepatocytes). In addition, rat albumin levels reached those of syngeneic recipients. Four months after transplantation of chimeric livers, we observed the development of diffuse mature rat bile ducts through transdifferentiation of hepatocytes (up to 72% of cholangiocytes), and patchy areas of portal endothelium originating from the host (seen in one out of five recipients). Taken together, these data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant recipient-oriented graft remodelling. Validation in a large animal model is still needed. Chimeric animals are composed of cells from different species. Chimeric animals carrying human tissue have the potential to increase the availability of transplantable organs. We transplanted rat-to-mouse liver grafts into newly weaned rats. The chimeric grafts underwent post-transplant remodelling with rat hepatocytes replacing all mouse hepatocytes within 56 days. In addition, we observed the post-transplant development of diffuse mature rat bile ducts through the transformation of hepatocytes, and patchy areas of portal endothelium originating from the host. These data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant graft remodelling.

Research Highlights

Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration Natarajan N, Abbas Y, Bryant DM, et al. Circulation. 2018;137:2152-2165. The organ shortage is perhaps the most pressing challenge facing the modern field of transplantation. Several approaches are being actively pursued to address this dilemma, including publicity drives to enhance donation rates, paired exchange donation, and an expanded use of marginal donor organs as well as their preconditioning. However, as the burden of organ failure increases, it is not entirely clear whether these approaches will fully address the organ shortage. Over the past decade, experimental techniques have focused on xenotransplantation and regenerative medicine to provide a supply of healthy organs or to repair endogenous organs. These approaches are now starting to gain traction as potentially realistic solutions1. In comparison to most organs, the heart has poor regenerative capabilities. Nevertheless, neonates in addition to some other animals, such as axolotl (also known as the Mexican Salamander) and zebrafish demonstrate enhanced cardiac regenerative ability in comparison to adult mammals. In a study from Natarajan and coworkers published in Circulation, a transcriptomic screen was performed across axolotl, zebrafish and mice to assess the molecular pathways that may be involved in their cardiac regenerative ability2. Ventricular apical resection was performed at various time points and RNA isolated from a section of the ventricle that remained in situ. RNA sequencing revealed an upregulation in inflammatory process genes. In particular, complement 5a receptor 1 (C5aR1) was found to be upregulated in all 3 species. In vivo inhibition of C5aR1 using a highly selective antagonist (PMX205) or genetic knockout significantly attenuated cardiomyocyte proliferation. C5aR1 is activated by complement 5a (C5a), and this pathway is an established innate immune activator. Interestingly, C5a has also previously been shown to be relevant in mouse liver regeneration3. Nevertheless, the authors found that treatment with C5a in vitro did not enhance cardiomyocyte proliferation, whereas in vivo treatment produced only a marginal increase in cardiomyocyte proliferation. Whether this pathway truly defines a specific therapeutic target remains unclear. However, the methodology in this article and the RNA-sequencing data (which has been deposited publicly) may reveal some of the mechanisms relevant to cardiac regeneration and provide useful insights for the regenerative medicine field. REFERENCES Pierson RN 3rd. Brief Summary Report From the 14th Biennial Meeting of the International Xenotransplantation Association. Transplantation. 2018;102:757–759. Natarajan N, Abbas Y, Bryant DM, et al. Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration. Circulation. 2018;137:2152–2165. Strey CW, Markiewski M, Mastellos D, et al. The proinflammatory mediators C3a and C5a are essential for liver regeneration. J Exp Med. 2003;198:913–923. De Novo Formation of the Biliary System by TGBβ-mediated Hepatocyte Transdifferentiation Schaub JR, Hupper KA, Kurial SNT, et al. [published online May 2, 2018]. Nature. DOI: 10.1038/s41586-018-0075-5. In contrast to stem cell–mediated regeneration, transdifferentiation is defined as a cellular plasticity that allows a change in cell identity as an alternative method for specific cellular replacement.1 An organ with inherent regenerative capabilities is the liver. In response to injury, hepatocytes undergo transdifferentiation to form biliary ductules, although it is thought that these do not form functional bile ducts, with ductiles reverting to a hepatocyte phenotype after injury resolution. A question therefore remains over whether this process is metaplasia rather than transdifferentiation, particularly because it is of unknown physiological relevance. In the second study in this month's Research Highlights, this question is addressed by Schaub and co-workers in the May 2018 issue of Nature.2 In contrast to previous studies, the study used a model where mice have severe cholestasis due to a lack of peripheral bile ducts, mimicking human Alagille syndrome, therefore allowing an accurate assessment of the functional capacity of transdifferentiated hepatocytes. Using fate-tracing assays, the authors found that hepatocytes do indeed differentiate into functional cholangiocytes that form de novo bile ducts which are functionally able to drain bile and persist after the liver injury is reversed. This hepatocyte plasticity therefore represents transdifferentiation rather than metaplasia. Through RNA sequencing gene ontology analysis, TGFβ signaling was found to be active in transdifferentiated cholangiocytes. Inhibition of TGFβ signaling prevented bile duct transdifferentiation, whereas activation of TGFβ signaling (using a specific vector) enhanced hepatocyte-derived bile duct formation. Interestingly, in liver samples derived from patients with Alagille syndrome, evidence for TGFβ signaling in peripheral bile ducts was confirmed by detection of nuclear pSMAD3. These mechanisms and the identification of TGFβ signaling as a potential therapeutic target represent fascinating leads for furthering our understanding the process of liver regeneration. REFERENCES Merrell AJ, Stanger BZ. Adult cell plasticity in vivo: de-differentiation and transdifferentiation are back in style. Nat Rev Mol Cell Biol. 2016;17:413–425. Schaub JR, Huppert KA, Kurial SNT, et al. De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation. [Published online May 2, 2018]. Nature. DOI:10.1038/s41586-018-0075-5.

De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation.

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, i.e., in the presence of a fully developed scaffold and niche1. Here we show that transdifferentiation of hepatocytes in the liver can build a structure that failed to form in development—the biliary system in mice that mimic the hepatic phenotype of human Alagille syndrome (ALGS)2. In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, i.e., incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system3–6. We show that, in contrast to bile duct development7–9, de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signaling. We identify TGFβ signaling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. We also show that TGFβ signaling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodeling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for therapy of ALGS and other cholestatic liver diseases.