Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson's disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Innovative application of an implantable venous access system in the portal vein: technique, results and complications in three dogs.

BACKGROUND: Vascular access port (VAP) systems are widely used in human medicine to provide long-term venous access. However, in veterinary medicine the use of VAP systems is not common practice and publications on their potential applications have been limited. A VAP system was used as part of an experimental study on liver regeneration and implanted in the canine portal vein to create direct access to the portal venous circulation of the liver. The aim of the present study is to describe the surgical technique, its use, and the complications of a VAP system in three research dogs. RESULTS: The VAP system was successfully used for the intraoperative measurement of portal blood pressure, the administration of cell suspensions, and the collection of portal venous blood samples. Long-term complications consisted of dislocation of the VAP system in one dog (2 months after implantation) and thrombus formation at the catheter tip in two dogs (3 months after implantation). Both complications prevented further use of the VAP but had no adverse clinical implications. CONCLUSIONS: This pilot study suggests that the VAP system is an effective and safe technique to obtain long term access to the portal venous system in dogs. However, complications with port detachment and thrombosis may limit long term use of VAPs in the portal system of dogs.

Immunohistochemical characterisation of the hepatic stem cell niche in feline hepatic lipidosis: a preliminary morphological study.

OBJECTIVES: The aim of this study was to describe the cellular and stromal components of the hepatic progenitor cell niche in feline hepatic lipidosis (FHL). METHODS: Immunohistochemical staining for the progenitor/bile duct marker (K19), activated Kupffer cells (MAC387), myofibroblasts (alpha-smooth muscle actin [α-SMA]) and the extracellular matrix component laminin were used on seven liver biopsies of cats with FHL and three healthy cats. Double immunofluorescence stainings were performed to investigate co-localisation of different cell types in the hepatic progenitor cell (HPC) niche. RESULTS: HPCs, Kupffer cells, myofibroblasts and laminin deposition were observed in the liver samples of FHL, although with variability in the expression and positivity of the different immunostainings between different samples. When compared with the unaffected cats where K19 positivity and minimal α-SMA and laminin positivity were seen mainly in the portal area, in the majority of FHL samples K19 and α-SMA-positive cells and laminin positivity were seen also in the periportal and parenchymatous area. MAC387-positive cells were present throughout the parenchyma. CONCLUSIONS AND RELEVANCE: This is a preliminary morphological study to describe the activation and co-localisation of components of the HPC niche in FHL. Although the HPC niche in FHL resembles that described in hepatopathies in dogs and in feline lymphocytic cholangitis, the expression of K19, α-SMA, MAC387 and lamin is more variable in FHL, and a common pattern of activation could not be established. Nevertheless, when HPCs were activated, a spatial association between HPCs and their niche could be demonstrated.

DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells.

Hepatic progenitor cells (HPCs) are adult liver stem cells that act as second line of defense in liver regeneration. They are normally quiescent, but in case of severe liver damage, HPC proliferation is triggered by external activation mechanisms from their niche. Although several important proproliferative mechanisms have been described, it is not known which key intracellular regulators govern the switch between HPC quiescence and active cell cycle. We performed a high-throughput kinome small interfering RNA (siRNA) screen in HepaRG cells, a HPC-like cell line, and evaluated the effect on proliferation with a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. One hit increased the percentage of EdU-positive cells after knockdown: dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Although upon DYRK1A silencing, the percentage of EdU- and phosphorylated histone H3 (pH3)-positive cells was increased, and total cell numbers were not increased, possibly through a subsequent delay in cell cycle progression. This phenotype was confirmed with chemical inhibition of DYRK1A using harmine and with primary HPCs cultured as liver organoids. DYRK1A inhibition impaired Dimerization Partner, RB-like, E2F, and multivulva class B (DREAM) complex formation in HPCs and abolished its transcriptional repression on cell cycle progression. To further analyze DYRK1A function in HPC proliferation, liver organoid cultures were established from mBACtgDyrk1A mice, which harbor one extra copy of the murine Dyrk1a gene (Dyrk+++). Dyrk+++ organoids had both a reduced percentage of EdU-positive cells and reduced proliferation compared with wild-type organoids. This study provides evidence for an essential role of DYRK1A as balanced regulator of S-phase entry in HPCs. An exact gene dosage is crucial, as both DYRK1A deficiency and overexpression affect HPC cell cycle progression.

Cellular characteristics of keratin 19-positive canine hepatocellular tumours explain its aggressive behaviour.

The expression of the hepatic progenitor cell marker keratin 19 (K19) in canine hepatocellular carcinomas is linked with a poor prognosis. To better understand this aggressive behaviour, K19-positive hepatocellular carcinomas (n=5) and K19-negative hepatocellular adenomas (n=6) were immunohistochemically stained for proteins involved in malignant tumour development. The K19-positive carcinomas showed marked positivity for platelet-derived growth factor receptor alpha polypeptide (PDGFRα), laminin, integrin beta-1/CD29, B-cell-specific Moloney murine leukaemia virus Integration site 1, glypican-3 (GPC-3) and prominin-1/CD133, in contrast with K19-negative hepatocellular adenomas. Conversely, neurofibromatosis type 2 was highly expressed in the hepatocellular adenomas in contrast with the hepatocellular carcinomas. This expression pattern is clearly in line with the observed aggressive behaviour. The presence of the malignancy markers PDGFRα and GPC-3 might make it possible to develop specific strategies to intervene in tumour growth and to devise novel serological tests and personalised treatment methods for canine hepatocellular carcinomas.

Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis.

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with ß-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research.

Concise Review: Organoids Are a Powerful Tool for the Study of Liver Disease and Personalized Treatment Design in Humans and Animals.

Organoids are three-dimensional culture systems in which adult stem cells and their progeny grow and represent the native physiology of the cells in vivo. Organoids have been successfully derived from several organ systems in both animal models and human patients. Organoids have been used for fundamental research, disease modeling, drug testing, and transplantation. In this review, we summarize the applications of liver-derived organoids and discuss their potential. It is likely that organoids will provide an invaluable tool to unravel disease mechanisms, design novel (personalized) treatment strategies, and generate autologous stem cells for gene editing and transplantation purposes.

Characterization and Comparison of Canine Multipotent Stromal Cells Derived from Liver and Bone Marrow.

Liver-derived multipotent stromal cells (L-MSCs) may prove preferable for treatment strategies of liver diseases, in comparison to the widely studied bone marrow-derived MSCs (BM-MSCs). Canines are a large animal model, in which the pathologies of liver diseases are similar to man. This study further promotes the implementation of canine models in MSC-based treatments of liver diseases. L-MSCs were characterized and compared to BM-MSCs from the same individual. Both cell types demonstrated a spindle-shaped fibroblast-like morphology, possessed the same growth potential, and demonstrated similar immunomodulation gene expression of CD274, PTGS-1, and PTGS-2. Marked differences in cell surface markers, CD105 and CD146, distinguished these two cell populations, and L-MSCs retained a liver-specific imprinting, observed by expression of CK18 and CK19. Finally, both populations differentiated toward the osteogenic and adipogenic lineage; however, L-MSCs failed to differentiate into the chondrogenic lineage. In conclusion, characterization of canine L-MSCs and BM-MSCs demonstrated that the two cell type populations are highly comparable. Although it is still unclear which cell source is preferred for clinical application in liver treatment strategies, this study provides a foundation for future controlled studies with MSC therapy in various liver diseases in dogs before their application in man.

Disease Modeling and Gene Therapy of Copper Storage Disease in Canine Hepatic Organoids.

The recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease.

Aberrant expression of copper associated genes after copper accumulation in COMMD1-deficient dogs.

BACKGROUND: COMMD1-deficient dogs progressively develop copper-induced chronic hepatitis. Since high copper leads to oxidative damage, we measured copper metabolism and oxidative stress related gene products during development of the disease. METHODS: Five COMMD1-deficient dogs were studied from 6 months of age over a period of five years. Every 6 months blood was analysed and liver biopsies were taken for routine histological evaluation (grading of hepatitis), rubeanic acid copper staining and quantitative copper analysis. Expression of genes involved in copper metabolism (COX17, CCS, ATOX1, MT1A, CP, ATP7A, ATP7B, ) and oxidative stress (SOD1, catalase, GPX1 ) was measured by qPCR. Due to a sudden death of two animals, the remaining three dogs were treated with d-penicillamine from 43 months of age till the end of the study. Presented data for time points 48, 54, and 60 months was descriptive only. RESULTS: A progressive trend from slight to marked hepatitis was observed at histology, which was clearly preceded by an increase in semi-quantitative copper levels starting at 12 months until 42 months of age. During the progression of hepatitis most gene products measured were transiently increased. Most prominent was the rapid increase in the copper binding gene product MT1A mRNA levels. This was followed by a transient increase in ATP7A and ATP7B mRNA levels. CONCLUSIONS: In the sequence of events, copper accumulation induced progressive hepatitis followed by a transient increase in gene products associated with intracellular copper trafficking and temporal activation of anti-oxidative stress mechanisms.

Hepatic progenitor cells in canine and feline medicine: potential for regenerative strategies.

New curative therapies for severe liver disease are urgently needed in both the human and veterinary clinic. It is important to find new treatment modalities which aim to compensate for the loss of parenchymal tissue and to repopulate the liver with healthy hepatocytes. A prime focus in regenerative medicine of the liver is the use of adult liver stem cells, or hepatic progenitor cells (HPCs), for functional recovery of liver disease. This review describes recent developments in HPC research in dog and cat and compares these findings to experimental rodent studies and human pathology. Specifically, the role of HPCs in liver regeneration, key components of the HPC niche, and HPC activation in specific types of canine and feline liver disease will be reviewed. Finally, the potential applications of HPCs in regenerative medicine of the liver are discussed and a potential role is suggested for dogs as first target species for HPC-based trials.

Enhanced Wnt/ß-catenin and Notch signalling in the activated canine hepatic progenitor cell niche.

BACKGROUND: The liver has a large regenerative capacity. Hepatocytes can replicate and regenerate a diseased liver. However, as is the case in severe liver diseases, this replication may become insufficient or exhausted and hepatic progenitor cells (HPCs) can be activated in an attempt to restore liver function. Due to their bi-potent differentiation capacity, these HPCs have great potential for regenerative approaches yet over-activation does pose potential health risks. Therefore the mechanisms leading to activation must be elucidated prior to safe implementation in the veterinary clinic. Wnt/ß-catenin and Notch signalling have been implicated in the activation of HPCs in mouse models and in humans. Here we assessed the involvement in canine HPC activation. Gene-expression profiles were derived from laser microdissected HPC niches from lobular dissecting hepatitis (LDH) and normal liver tissue, with a focus on Wnt/ß-catenin and Notch signalling. Immunohistochemical and immunofluorescent studies were combined to assess the role of the pathways in HPCs during LDH. RESULTS: Gene-expression confirmed higher expression of Wnt/ß-catenin and Notch pathway components and target genes in activated HPC niches in diseased liver compared to quiescent HPC niches from normal liver. Immunofluorescence confirmed the activation of these pathways in the HPCs during disease. Immunohistochemistry showed proliferating HPCs during LDH, and double immunofluorescence showed downregulation of Wnt/ß-catenin and Notch in differentiating HPCs. Vimentin, a mesenchymal marker, was expressed on a subset of undifferentiated HPCs. CONCLUSIONS: Together these studies clearly revealed that both Wnt/ß-catenin and Notch signalling pathways are enhanced in undifferentiated, proliferating and potentially migrating HPCs during severe progressive canine liver disease (LDH).

Potential of regenerative medicine techniques in canine hepatology.

Liver cell turnover is very slow, especially compared to intestines and stomach epithelium and hair cells. Since the liver is the main detoxifying organ in the body, it does not come as a surprise that the liver has an unmatched regenerative capacity. After 70% partial hepatectomy, the liver size returns to normal in about two weeks due to replication of differentiated hepatocytes and cholangiocytes. Despite this, liver diseases are regularly encountered in the veterinary clinic. Dogs primarily present with parenchymal pathologies such as hepatitis. The estimated frequency of canine hepatitis depends on the investigated population and accounts for 1%-2% of our university clinic referral population, and up to 12% in a general population. In chronic and severe acute liver disease, the regenerative and replicative capacity of the hepatocytes and/or cholangiocytes falls short and the liver is not restored. In this situation, proliferation of hepatic stem cells or hepatic progenitor cells (HPCs), on histology called the ductular reaction, comes into play to replace the damaged hepatocytes or cholangiocytes. For unknown reasons the ductular reaction is often too little and too late, or differentiation into fully differentiated hepatocytes or cholangiocytes is hampered. In this way, HPCs fail to fully regenerate the liver. The presence and potential of HPCs does, however, provide great prospectives for their use in regenerative strategies. This review highlights the regulation of, and the interaction between, HPCs and other liver cell types and discusses potential regenerative medicine-oriented strategies in canine hepatitis, making use of (liver) stem cells.

COMMD1-deficient dogs accumulate copper in hepatocytes and provide a good model for chronic hepatitis and fibrosis.

New therapeutic concepts developed in rodent models should ideally be evaluated in large animal models prior to human clinical application. COMMD1-deficiency in dogs leads to hepatic copper accumulation and chronic hepatitis representing a Wilson's disease like phenotype. Detailed understanding of the pathogenesis and time course of this animal model is required to test its feasibility as a large animal model for chronic hepatitis. In addition to mouse models, true longitudinal studies are possible due to the size of these dogs permitting detailed analysis of the sequence of events from initial insult to final cirrhosis. Therefore, liver biopsies were taken each half year from five new born COMMD1-deficient dogs over a period of 42 months. Biopsies were used for H&E, reticulin, and rubeanic acid (copper) staining. Immunohistochemistry was performed on hepatic stellate cell (HSC) activation marker (alpha-smooth muscle actin, α-SMA), proliferation (Ki67), apoptosis (caspase-3), and bile duct and liver progenitor cell (LPC) markers keratin (K) 19 and 7. Quantitative RT-PCR and Western Blots were performed on gene products involved in the regenerative and fibrotic pathways. Maximum copper accumulation was reached at 12 months of age, which coincided with the first signs of hepatitis. HSCs were activated (α-SMA) from 18 months onwards, with increasing reticulin deposition and hepatocytic proliferation in later stages. Hepatitis and caspase-3 activity (first noticed at 18 months) increased over time. Both HGF and TGF-ß1 gene expression peaked at 24 months, and thereafter decreased gradually. Both STAT3 and c-MET showed an increased time-dependent activation. Smad2/3 phosphorylation, indicative for fibrogenesis, was present at all time-points. COMMD1-deficient dogs develop chronic liver disease and cirrhosis comparable to human chronic hepatitis, although at much higher pace. Therefore they represent a genetically-defined large animal model to test clinical applicability of new therapeutics developed in rodent models.

Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.

BACKGROUND: Hepatic progenitor cells (HPCs) hold a great potential for therapeutic intervention for currently untreatable liver diseases. However, in human diseases molecular mechanisms involved in proliferation and differentiation of HPCs are poorly understood. METHODS AND RESULTS: In the present study activated HPCs and their microenvironment (niche) were investigated in acute and chronic human liver disease by gene-expression analysis and immunohistochemistry/immunofluorescence. Cryopreserved liver tissues were used from patients with parenchymal versus biliary diseases: acute necrotising hepatitis (AH), cirrhosis after hepatitis C infection, and primary biliary cirrhosis in order to study differentiation of HPCs towards hepatocytic versus biliary lineage. Keratin 7 positive HPCs/reactive ductules were captured by means of laser capture microdissection and gene-expression profiles were obtained by using a customized PCR array. Gene expression results were confirmed by immunohistochemistry and immunofluorescence double staining. In all disease groups, microdissected HPCs expressed progenitor cell markers such as KRT7, KRT19, NCAM, ABCG2, LIF, KIT, OCT4, CD44 and TERT. In AH, HPCs were most activated and showed a high expression of prominin-1 (CD133) and alpha-fetoprotein, and a strong activation of the Wnt pathway. In contrast to parenchymal diseases, HPCs in primary biliary cirrhosis (biliary differentiation) showed a high activation of Notch signalling. CONCLUSION: A distinct pattern of HPC surface markers was found between acute and chronic liver diseases. Similar to what is known from animal experiments, strong evidence has been found signifying the role of Wnt signalling in proliferation of human HPCs whereas Notch signalling is involved in biliary differentiation. These pathways can be targeted in future therapies.

Comparison of different methods to obtain and store liver biopsies for molecular and histological research.

BACKGROUND: To minimize the necessary number of biopsies for molecular and histological research we evaluated different sampling techniques, fixation methods, and storage procedures for canine liver tissue. For addressing the aim, three biopsy techniques (wedge biopsy, Menghini, True-cut), four storage methods for retrieval of RNA (snap freezing, RNAlater, Boonfix, RLT-buffer), two RNA isolation procedures (Trizol and RNAeasy), and three different fixation protocols for histological studies (10% buffered formalin, RNAlater, Boonfix) were compared. Histological evaluation was based on hematoxylin-eosin (HE) and reticulin (fibrogenesis) staining, and rubeanic acid and rhodanine stains for copper. Immunohistochemical evaluation was performed for cytokeratin-7 (K-7), multidrug resistance binding protein-2 (MRP-2) and Hepar-1. RESULTS: RNA quality was best guaranteed by the combination of a Menghini biopsy with NaCl, followed by RNAlater preservation and RNAeasy mini kit extraction. These results were confirmed by quantitative RT-PCR testing. Reliable histological assessment for copper proved only possible in formalin fixed liver tissue. Short formalin fixation (1-4 hrs) improved immunohistochemical reactivity and preservation of good morphology in small liver biopsies. CONCLUSION: At least two biopsies (RNAlater and formalin) are needed. Since human and canine liver diseases are highly comparable, it is conceivable that the protocols described here can be easily translated into the human biomedical field.

Cross-species immunohistochemical investigation of the activation of the liver progenitor cell niche in different types of liver disease.

BACKGROUND: When hepatocyte replication during liver disease is insufficient for regeneration, liver progenitor cells (LPCs) are activated. The cells and stroma in the immediate environment of LPCs, together termed the LPC niche, are thought to play an important role in this activation. Among these cells are the hepatic stellate cells (HSCs)/myofibroblasts (MFs). AIMS/METHODS: We assessed the activation of HSC/MFs and LPCs in relation to the histological location and extent of liver disease in immunohistochemically (double) stained serial sections. Markers of HSC/MFs [alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP), neurotrophin 3 and neural-cell adhesion molecule], markers of LPCs (keratin 7 and keratin 19) and a proliferation marker (Ki67) were used. A very relevant spontaneous model to evaluate LPC niche activation in a translational approach seems to be the dog. Therefore, both human and canine liver diseases with different degree of fibrosis and disease activity were included. RESULTS: In human and canine liver disease, type and extent of LPC niche activation depended on type and severity of disease (P<0.05) and corresponded to the main location of disease. Activated HSCs surrounded the activated LPCs. In chronic hepatitis and non-alcoholic steatohepatitis lobular-type HSCs were activated, while during biliary disease portal/septal MFs were mainly activated. In canine liver, GFAP further presented as an early marker of HSC activation. Activation of the LPCs correlated with disease location and severity (P<0.01), and was inversely related to hepatocyte proliferation, as was previously shown in man. CONCLUSION: A shared involvement of HSC/MFs, LPCs and disease severity during hepatic disease processes is shown, which is highly similar in man and dog.

In vitro differentiation of liver progenitor cells derived from healthy dog livers.

Naturally occurring liver disease in dogs resemble human liver disease in great detail; including the activation of liver progenitor cells (LPC) in acute and chronic liver disease. The aim of the present study was to isolate, culture, and characterize progenitor cells derived from healthy mature dog livers. A nonparenchymal cell fraction enriched with small hepatocytes was isolated and cultured in Hepatozyme-serum-free media (SFM) to stimulate the growth of colony-forming small epithelial cells. After 2 weeks of culturing, clonal expansion of keratin 7 (K7) immunopositive small cells with a large nucleus/cytoplasm ratio emerged in the hepatocyte monolayer. These colonies expressed genes of several hepatocyte (CYP1A1, ALB, and KRT18), cholangiocyte/LPC (KRT7 and KRT19), and progenitor cell markers (alpha-fetoprotein, CD44, prominin1, KIT, THY1, and neural cell adhesion molecule 1), indicating their immature and bipotential nature. Gene-expression profiles indicated a more pronounced hepatic differentiation in Hepatozyme-SFM compared to William's Medium E (WME). Furthermore, colony-forming cells differentiated toward intermediate hepatocyte-like cells with a more pronounced membranous K7 immunostaining. In conclusion, colony-forming small epithelial cells in long-term canine liver cell cultures express LPC markers and have differentiating capacities. These cells may therefore be considered as progenitor cells of the liver.