Cholangiocyte‐to‐Hepatocyte Differentiation: A Context‐Dependent Process and an Opportunity for Regenerative Medicine
Authors: Maria J. Perugorria, Paula Olaizola, Jesus M. Banales
Journal: Hepatology (2018)

Supported by the Spanish Ministries of Economy and Competitiveness (J.M.B. [FIS PI12/00380, FIS PI15/01132, PI18/01075 and Miguel Servet Programme CON14/00129]; M.J.P. [FIS PI14/00399, FIS PI17/00022] and Ramon y Cajal Programme RYC‐2015‐17755) cofinanced by "Fondo Europeo de Desarrollo Regional" (FEDER); ISCIII (CIBERehd: J.M.B. and M.J.P.), Spain; "Diputación Foral de Gipuzkoa" ([J.M.B.: DFG15/010, DFG16/004]; M.J.P.: DFG1818/114]); BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD to J.M.B.); Department of Health of the Basque Country (J.M.B.: 2017111010; M.J.P.: 2015111100), and AECC Scientific Foundation (J.M.B.). P.O. was funded by the Basque Government (PRE_2016_1_0152). SEE ARTICLE ON PAGE 742 The liver possesses an extraordinary regenerative capacity to compensate functional mass loss after acute cytotoxic injury or surgical resection. Hepatic mass restoration following hepatocellular or cholangiocellular injury is commonly mediated by the proliferation and replenishment of the existing differentiated hepatocytes and biliary epithelial cells (BECs or cholangiocytes), respectively. However, during chronic hepatocellular damage, the long‐term cell renewal capacity of differentiated hepatocytes becomes compromised. Under these circumstances, hepatocytes can enter into replicative senescence, leading to the exhaustion of their proliferative ability, and, therefore, are no longer able to regenerate the hepatic mass. Nevertheless, patients with chronic liver injury exhibit ductular reaction with increased levels of replicating BECs, which correlate with advanced stages of the disease.1 In this regard, the contribution of the pluripotent BECs, derived from the small ducts of the intrahepatic biliary tree (i.e., canals of Hering), to hepatocyte mass restoration was suggested. Although the first studies reported a modest involvement of BECs in hepatocyte repopulation,2 recent research work has demonstrated the capacity of BECs to transdifferentiate into hepatocytes upon impaired hepatocyte proliferation. Indeed, cholangiocyte‐to‐hepatocyte transdifferentiation has been demonstrated in both human3 and animal models of liver injury characterized by reduced hepatocyte expansion/proliferation,4 senescence,5 or extended exposure to liver‐damaging agents.6 Different animal models have been used to study the activation and proliferation of reactive BECs, including the choline‐deficient ethionine‐supplemented (CDE) diet, 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) diet, methionine‐ and choline‐deficient (MCD) diet, and thioacetamide (TAA). First evidences using lineage‐tracing studies in CDE diet–fed mice concluded that BECs were not a major source of new hepatocytes and significant contributors of hepatocyte mass restoration.2 However, recent studies in transgenic mouse models of widespread hepatocyte injury and senescence,5 or inhibition of hepatocyte proliferation,4 have demonstrated that reactive BECs act as stem cells during impaired liver regeneration and significantly contribute to hepatocyte replacement. This phenomenon was reproduced with different diet regimens, including CDE, DDC, MCD, and TAA.4 Of note, with the aim of modeling human chronic liver injury, a recent study carried out in mice with severe liver injury following long‐term thioacetamide or DDC treatment and without any genetic intervention, has further supported conversion of BECs into hepatocytes.6 These novel results are encouraging but need to be validated and expanded, adding new information regarding the context of this regenerative process, the molecular mechanisms involved, and the therapeutic opportunities that offers. In this issue of hepatology, Russell et al.7 have investigated BEC‐to‐hepatocyte transdifferentiation, performing sophisticated lineage‐tracing studies in conditional β‐catenin knockout mouse models following CDE diet–induced injury, with the aim of studying the potential contribution of BECs to the restoration of hepatocyte mass after chronic liver injury when hepatocyte proliferation is impaired. The Wnt/β‐catenin signaling pathway is fundamental in liver development, health, disease, and regeneration.8 Among others, this molecular pathway promotes hepatocyte proliferation, and its defects result in delayed liver regeneration.9 In order to investigate whether the liver‐specific loss of β‐catenin expression in combination with CDE diet–induced liver injury impairs hepatocyte proliferation, and this triggers ductular reaction, authors used Albumin‐Cre Ctnnb1 knock‐out (KO) mice, which lack β‐catenin expression in both hepatocytes and BECs. Notably, chronic (2 weeks) feeding of Albumin‐Cre Ctnnb1 KO mice with CDE diet resulted in severe liver injury, impaired hepatocyte proliferation, and a robust BEC expansion, despite β‐catenin loss in these cells. These data confirm the role of β‐catenin as a pivotal regulator of the regenerative capacity of hepatocytes after liver injury, and further point out the existence of an associated β‐catenin independent ductular reaction. Additionally, a hepatocyte‐specific β‐catenin KO mouse model (Ctnnb1flox/flox;Rosa‐stopflox/flox‐EYFP;TBG‐Cre) was generated by injecting these mice with an adeno‐associated virus encoding Cre recombinase under thyroid‐binding globulin promoter (AAV8‐TBG‐Cre) to permanently label β‐catenin KO hepatocytes with enhanced yellow fluorescence protein (EYFP). Feeding these mice with CDE diet for 2 weeks resulted in severe liver injury, nearly absent hepatocyte proliferation, and no BEC‐derived hepatocytes. However, when the 2‐week period of CDE diet was followed by 2 weeks of normal diet, almost 20% of hepatocyte nuclear factor 4 α (Hnf4α)‐positive hepatocytes were β‐catenin positive and EYFP negative, demonstrating that under a recovery period, reactive BECs can transdifferentiate into hepatocytes. Most of these BEC‐derived hepatocytes did not express cholangiocyte markers, were clustered in the periportal region neighboring BECs, and exhibited increased proliferation, indicating their potential role in repopulating the hepatic parenchymal loss. In particular, β‐catenin+/Hnf4α+/cytokeratin 19+ hepatocyte‐like cells (with hepatocyte morphology) were observed just 3 days after the administration of a normal diet, showing that the transdifferentiation of BECs is a dynamic process that occurs over time. Of note, Russel et al. demonstrated that this BEC‐to‐hepatocyte conversion is permanent in long‐term recovery studies, in which approximately 70% of the hepatocytes, defined as Hnf4α‐positive cells, are EYFP negative after 3 months of recovery, and their numbers remain constant after 6 months. Supporting these findings, authors performed BEC lineage‐tracing studies using Krt19‐CreERT;Rosa‐stopflox/flox;tdTomato labelling BECs and simultaneously knocking down Ctnnb1 in hepatocytes with GalXC‐Ctnnb1, a Ctnnb1 RNAi conjugated to hepatocyte‐targeting ligand N‐acetylgalactosamine, to induce hepatocyte‐specific knockdown of β‐catenin. In line with previous observations, Ctnnb1 knockdown in hepatocytes inhibited their proliferative capacity and induced severe liver injury, promoting the appearance of BEC‐derived hepatocytes during the recovery phase, contributing to the restoration of the lost parenchymal mass. Overall, these results contribute to expand the knowledge on the plasticity of BECs, and demonstrate their capacity to proliferate and transdifferentiate to hepatocytes after liver injury and impaired hepatocyte proliferation, as well as their long‐term liver repopulation capacity. This study mechanistically demonstrates the important role of β‐catenin in the regulation of the regenerative capacity of hepatocytes, and identified a β‐catenin independent BEC expansion. However, the presence of active β‐catenin signaling observed in Ctnnb1flox/flox;Rosa‐stopflox/flox‐EYFP;TBG‐Cre mice livers suggests that β‐catenin could play a potential role in BEC‐to‐hepatocyte differentiation, as previously suggested.10 Nonetheless, the presence of active β‐catenin expression in other nonparenchymal cells cannot be ruled out in this model and needs to be further elucidated. The fact that the transdifferentiation process in the current study was only observed once the damaging agent was removed and after a recovery phase is of great relevance. In fact, this cellular event could participate in the regeneration of the liver in humans with chronic liver disease who experience liver injury resolution or are no longer exposed to the primary etiologic factor. For instance, this could occur in chronic hepatitis C virus–infected patients after antiviral treatment, obese patients with nonalcoholic steatohepatitis after lifestyle modifications, or patients with alcoholic steatohepatitis once alcohol consumption ceases. Furthermore, elucidating the molecular mechanisms driving the conversion of BECs into hepatocytes would provide new therapeutic opportunities in regenerative medicine to handle chronic liver diseases. Nevertheless, the hepatic damage threshold triggering this differentiation and the subsequent mass replenishment, as well as the functionality of these cells and their long‐term effects in the liver, remain to be determined. In conclusion (Fig. 1), the study by Russell et al. nicely demonstrates that reactive BECs can transdifferentiate into hepatocytes, at least under certain experimental conditions of chronic liver disease, contributing to organ repopulation. Because the only current therapeutic option for late‐stage chronic liver diseases is liver transplantation, which represents a challenge because of the scarcity of donor organs, this study opens new avenues for future therapeutic interventions based on regenerative medicine.Figure 1: Hepatocyte‐ and cholangiocyte‐mediated regenerative response to (A) acute and (B) chronic liver injury. Abbreviations: BEC, biliary epithelial cell; LSEC, liver sinusoidal endothelial cell.Potential conflict of interest Nothing to report. Author names in bold designate shared co‐first authorship.