Locational memory of macrovessel vascular cells is transcriptionally imprinted.

Vascular pathologies show locational predisposition throughout the body; further insights into the transcriptomics basis of this vascular heterogeneity are needed. We analyzed transcriptomes from cultured endothelial cells and vascular smooth muscle cells from nine adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We observed that organ-specific expression patterns persist in vitro, indicating that these genes are not regulated by blood flow or surrounding cell types but are likely fixed in the epigenetic memory. We further demonstrated the preserved location-specific expression of GATA4 protein in cultured cells and in the primary adult vessel. On a functional level, arterial and venous endothelial cells differed in vascular network morphology as the arterial networks maintained a higher complexity. Our findings prompt the rethinking of the extrapolation of results from single-origin endothelial cell systems.

Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.

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.

Gene expressions of de novo hepatic lipogenesis in feline hepatic lipidosis.

OBJECTIVES: The aim of this study was to evaluate if de novo hepatic lipid synthesis contributes to fatty acid overload in the liver of cats with feline hepatic lipidosis (FHL). METHODS: Lipogenic gene expression of peroxisome proliferator-activated receptor-alpha (PPAR-α), peroxisome proliferator-activated receptor-gamma (PPAR-γ), fatty acid synthase (FASN) and sterol regulatory element-binding factor (SREBF1) were evaluated using quantitative RT-PCR in liver tissue of six cats with FHL and compared with the liver tissue of eight healthy cats. RESULTS: In liver tissue, PPAR-α, PPAR-γ and FASN mRNA expression levels were not significantly different (P >0.12, P >0.89 and P >0.5, respectively) in the FHL group compared with the control group. SREBF1 gene expression was downregulated around 10-fold in the FHL group vs the control group (P = 0.039). CONCLUSIONS AND RELEVANCE: The downregulation of SREBF1 in the liver tissue of cats with FHL does not support the hypothesis that de novo lipogenesis in the liver is an important pathway of fatty acid accumulation in FHL.

Identification of potential drugs for treatment of hepatic lipidosis in cats using an in vitro feline liver organoid system.

BACKGROUND: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver. OBJECTIVE: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. ANIMALS: Liver organoids obtained from 6 cats. METHODS: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. RESULTS: Both T863 (a diacylglycerol O-acyltransferase 1 [DGAT1] inhibitor) and 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P < .0001) and 46% (P = .0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863. CONCLUSIONS AND CLINICAL IMPORTANCE: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid-lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species-specific system and provide the basis for further clinical testing of drugs to treat steatosis.

Reduced FXR Target Gene Expression in Copper-Laden Livers of COMMD1-Deficient Dogs.

Wilson's disease (WD), an autosomal recessive disorder, results in copper accumulation in the liver as a consequence of mutations in the gene ATPase copper transporting beta (ATP7B). The disease is characterized by chronic hepatitis, eventually resulting in liver cirrhosis. Recent studies have shown that dysregulation of nuclear receptors (NR) by high hepatic copper levels is an important event in the pathogenesis of liver disease in WD. Intracellular trafficking of ATP7B is mediated by COMMD1 and, in Bedlington terriers, a mutation in the COMMD1 gene results in high hepatic copper levels. Here, we demonstrate a reduced Farnesoid X nuclear receptor (FXR)-activity in liver biopsies of COMMD1-deficient dogs with copper toxicosis, a unique large animal model of WD. FXR-induced target genes, small heterodimer partner (SHP), and apolipoprotein E (ApoE) were down-regulated in liver samples from COMMD1-deficient dogs with hepatic copper accumulation. In contrast, the relative mRNA levels of the two CYP-enzymes (reduced by FXR activity) was similar in both groups. These data are in line with the previously observed reduced FXR activity in livers of ATP7B-/- mice and WD patients. Therefore, these data further corroborate on the importance of the COMMD1-deficient dogs as a large animal model for WD.

Towards Bioengineered Liver Stem Cell Transplantation Studies in a Preclinical Dog Model for Inherited Copper Toxicosis.

Wilson Disease is a rare autosomal recessive liver disorder in humans. Although its clinical presentation and age of onset are highly variable, hallmarks include signs of liver disease, neurological features and so-called Kayser-Fleischer rings in the eyes of the patient. Hepatic copper accumulation leads to liver disease and eventually to liver cirrhosis. Treatment options include life-long copper chelation therapy and/or decrease in copper intake. Eventually liver transplantations are indicated. Although clinical outcome of liver transplantations is favorable, the lack of suitable donor livers hampers large numbers of transplantations. As an alternative, cell therapies with hepatocytes or liver stem cells are currently under investigation. Stem cell biology in relation to pets is in its infancy. Due to the specific population structure of dogs, canine copper toxicosis is frequently encountered in various dog breeds. Since the histology and clinical presentation resemble Wilson Disease, we combined genetics, gene-editing, and matrices-based stem cell cultures to develop a translational preclinical transplantation model for inherited copper toxicosis in dogs. Here we describe the roadmap followed, starting from the discovery of a causative copper toxicosis mutation in a specific dog breed and culminating in transplantation of genetically-engineered autologous liver stem cells.

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.

Preclinical models of Wilson's disease, why dogs are catchy alternatives.

Copper toxicosis is frequently encountered in various dog breeds. A number of differences and similarities occur between Wilson disease and copper toxicosis in Bedlington terriers, caused by a mutation in the COMMD1 gene, and copper toxicosis in Labrador retrievers, caused by mutations in both ATP7A and ATP7B gene. First the specific population structure of dog breeds is explained with reference to its applicability for genetic investigations. The relatively large body size (variable from less than 1 kg to over 50 kg) and life-span (over 10 years) of dogs facilitates preclinical studies on safety on long-term effects of novel procedures. Then copper toxicosis in the two dog breeds is described in detail with an emphasis on the functions of the causative proteins. Some of the advantages of this species for preclinical studies are described with an example of liver stem cell transplantations in COMMD1 deficient dogs. Since the genetic background of copper toxicosis in other dogs' breeds has not yet been elucidated, it is conceivable that novel copper-related gene products or modifier genes will be discovered. About a century after the Novel prize was awarded to the research on dogs (Pavlov), dogs are in spotlight again as important preclinical model animals.

Characterization of Endothelial and Smooth Muscle Cells From Different Canine Vessels.

Vasculature performs a critical function in tissue homeostasis, supply of oxygen and nutrients, and the removal of metabolic waste products. Vascular problems are implicated in a large variety of pathologies and accurate in vitro models resembling native vasculature are of great importance. Unfortunately, existing in vitro models do not sufficiently reflect their in vivo counterpart. The complexity of vasculature requires the examination of multiple cell types including endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as vessel location in the body from which they originate. The use of canine blood vessels provides a way to study vasculature with similar vessel size and physiology compared to human vasculature. We report an isolation procedure that provides the possibility to isolate both the endothelial and smooth muscle cells from the same vessels simultaneously, enabling new opportunities in investigating vasculature behavior. Canine primary ECs and VSMCs were isolated from the vena cava, vena porta and aorta. All tissue sources were derived from three donors for accurate comparison and to reduce inter-animal variation. The isolation and purification of the two distinct cell types was confirmed by morphology, gene- and protein-expression and function. As both cell types can be derived from the same vessel, this approach allows accurate modeling of vascular diseases and can also be used more widely, for example, in vascular bioreactors and tissue engineering designs. Additionally, we identified several new genes that were highly expressed in canine ECs, which may become candidate genes for novel EC markers. In addition, we observed transcriptional and functional differences between arterial- and venous-derived endothelium. Further exploration of the transcriptome and physiology of arteriovenous differentiation of primary cells may have important implications for a better understanding of the fundamental behavior of the vasculature and pathogenesis of vascular disease.

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.

Aberrant hepatic lipid storage and metabolism in canine portosystemic shunts.

Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS P<0.01; IHPSS P = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.

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.

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Intestinal Organoids-Current and Future Applications.

Recent technical advances in the stem cell field have enabled the in vitro generation of complex structures resembling whole organs termed organoids. Most of these approaches employ culture systems that allow stem cell-derived or tissue progenitor cells to self-organize into three-dimensional (3D)-structures. Since organoids can be grown from different species (human, mouse, cat, dog), organs (intestine, kidney, brain, liver), and from patient-derived induced pluripotent stem cells, they create significant prospects for modelling development and diseases, for toxicology and drug discovery studies, and in the field of regenerative medicine. Here, we report on intestinal stem cells, organoid culture, organoid disease modeling, transplantation, specifically covering the current and future uses of this exciting new insight model to the field of veterinary medicine.

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.

Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. We previously observed that the levels of triacylglycerol (TAG) species containing long polyunsaturated fatty acids (PUFAs) are increased in in vitro activated HSCs. Here we investigated the cause and consequences of the rise in PUFA-TAGs by profiling enzymes involved in PUFA incorporation. We report that acyl CoA synthetase (ACSL) type 4, which has a preference for PUFAs, is the only upregulated ACSL family member in activated HSCs. Inhibition of the activity of ACSL4 by siRNA-mediated knockdown or addition of rosiglitazone specifically inhibited the incorporation of deuterated arachidonic acid (AA-d8) into TAG in HSCs. In agreement with this, ACSL4 was found to be partially localized around lipid droplets (LDs) in HSCs. Inhibition of ACSL4 also prevented the large increase in PUFA-TAGs in HSCs upon activation and to a lesser extent the increase of arachidonate-containing phosphatidylcholine species. Inhibition of ACSL4 by rosiglitazone was associated with an inhibition of HSC activation and prostaglandin secretion. Our combined data show that upregulation of ACSL4 is responsible for the increase in PUFA-TAG species during activation of HSCs, which may serve to protect cells against a shortage of PUFAs required for eicosanoid secretion.

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).

Recombinant hepatocyte growth factor treatment in a canine model of congenital liver hypoplasia.

BACKGROUND: Although the liver has a large regenerative capacity, in many hepatopathies, these repair mechanisms fail. The therapeutic potential of hepatocyte growth factor (HGF) has been proven in numerous toxin-induced liver failure models in rodents, but never in spontaneously occurring liver diseases in larger animal models. AIM: The aim of this study was to induce liver growth in a hypoplastic liver by the administration of exogenous recombinant HGF. The natural hypoplastic liver model used is the canine congenital portosystemic shunt (CPSS) characterized by strongly reduced liver growth and function. METHODS: Recombinant HGF (rHGF), 200 µg/kg, was given twice daily during 3 weeks by an intravenous injection in six dogs with CPSS. Liver volumes were determined by computed tomography before and at 1, 2, 3 and 7 weeks after the initiation of treatment. Portosystemic shunting was evaluated with an ammonia tolerance test and liver portal perfusion was quantified with scintigraphy. Simultaneously, blood parameters for liver function were assayed and liver biopsies were taken for histology, immunohistochemistry and gene-expression measurements. RESULTS: During 3 weeks of HGF treatment, hepatocyte proliferation increased and an increase in liver volume up to 44% was seen, persisting in two dogs up to 4 weeks after the termination of treatment. Ki-67 expression, gene expression of E2F1 and CDC6, phosphorylated-c-MET and phosphorylated-ERK1/2 protein levels confirmed increased hepatocyte proliferation and HGF signalling. The aberrant portal perfusion did not change during treatment. CONCLUSIONS: Transient in vivo liver growth is shown using CPPS as a naturally occurring large animal model, indicating the therapeutic potential of HGF in liver disease.