High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

BACKGROUND AND AIMS: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

A Chemically Defined Hydrogel for Human Liver Organoid Culture.

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is poorly defined, suffers from high batch-to-batch variability and is of xenogeneic origin, which limits the clinical application of organoids. Here, a novel hydrogel based on polyisocyanopeptides (PIC) and laminin-111 is described for human liver organoid cultures. PIC is a synthetic polymer that can form a hydrogel with thermosensitive properties, making it easy to handle and very attractive for clinical applications. Organoids in an optimized PIC hydrogel proliferate at rates comparable to those observed with Matrigel; proliferation rates are stiffness-dependent, with lower stiffnesses being optimal for organoid proliferation. Moreover, organoids can be efficiently differentiated toward a hepatocyte-like phenotype with key liver functions. This proliferation and differentiation potential maintain over at least 14 passages. The results indicate that PIC is very promising for human liver organoid culture and has the potential to be used in a variety of clinical applications including cell therapy and tissue engineering.

Hepatitis E virus seroprevalence in pets in the Netherlands and the permissiveness of canine liver cells to the infection.

Hepatitis E virus (HEV) as an emerging zoonotic pathogen causes a major public health issue. Transmission from domestic, wildlife and zoo animals to human has been widely reported. Whether pets also serve as reservoirs remains an intriguing question. In this study, we found the sero-positive rates of HEV-specific antibodies in pet dogs, cats and horses of 18.52% (30/162), 14.89% (7/47) and 18.18% (4/22) in the Netherlands. Although HEV viral RNA was not detected in these animals, we have demonstrated that dog liver cells are susceptible to HEV infection in vitro. These results call more attention to address the potential role of pets in the zoonotic transmission of HEV.

Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers.

Direct reprogramming represents an easy technique to generate induced hepatocytes (iHeps) from somatic cells. However, current protocols are accompanied by several drawbacks as iHeps are heterogenous and lack fully mature phenotypes of primary hepatocytes. Here, we established a polycistronic expression system to induce the direct reprogramming of mouse embryonic fibroblasts towards hepatocytes. The resulting iHeps are homogenous and display key properties of primary hepatocytes, such as expression of hepatocyte markers, albumin secretion, and presence of liver transaminases. iHeps also possess the capacity to repopulate decellularized liver tissue and exhibit enhanced hepatic maturation. As such, we present a novel strategy to generate homogenous and functional iHeps for applications in tissue engineering and cell therapy.

The paracrine feedback loop between vitamin D3 (1,25(OH)2D3) and PTHrP in prehypertrophic chondrocytes.

The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.

Potential regenerative treatment strategies for intervertebral disc degeneration in dogs.

Pain due to spontaneous intervertebral disc (IVD) disease is common in dogs. In chondrodystrophic (CD) dogs, IVD disease typically develops in the cervical or thoracolumbar spine at about 3-7 years of age, whereas in non-chondrodystrophic (NCD) dogs, it usually develops in the caudal cervical or lumbosacral spine at about 6-8 years of age. IVD degeneration is characterized by changes in the biochemical composition and mechanical integrity of the IVD. In the degenerated IVD, the content of glycosaminoglycan (GAG, a proteoglycan side chain) decreases and that of denatured collagen increases. Dehydration leads to tearing of the annulus fibrosus (AF) and/or disc herniation, which is clinically characterized by pain and/or neurological signs. Current treatments (physiotherapy, anti-inflammatory/analgesic medication, surgery) for IVD disease may resolve neurological deficits and reduce pain (although in many cases insufficient), but do not lead to repair of the degenerated disc. For this reason, there is interest in new regenerative therapies that can repair the degenerated disc matrix, resulting in restoration of the biomechanical function of the IVD. CD dogs are considered a suitable animal model for human IVD degeneration because of their spontaneous IVD degeneration, and therefore studies investigating cell-, growth factor-, and/or gene therapy-based regenerative therapies with this model provide information relevant to both human and canine patients. The aim of this article is to review potential regenerative treatment strategies for canine IVD degeneration, with specific emphasis on cell-based strategies.

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).

Mice with renal-specific alterations of stem cell-associated signaling develop symptoms of chronic kidney disease but surprisingly no tumors.

Previously, we found that Wnt and Notch signaling govern stem cells of clear cell kidney cancer (ccRCC) in patients. To mimic stem cell responses in the normal kidney in vitro in a marker-unbiased fashion, we have established tubular organoids (tubuloids) from total single adult mouse kidney epithelial cells in Matrigel and serum-free conditions. Deep proteomic and phosphoproteomic analyses revealed that tubuloids resembled renewal of adult kidney tubular epithelia, since tubuloid cells displayed activity of Wnt and Notch signaling, long-term proliferation and expression of markers of proximal and distal nephron lineages. In our wish to model stem cell-derived human ccRCC, we have generated two types of genetic double kidney mutants in mice: Wnt-ß-catenin-GOF together with Notch-GOF and Wnt-ß-catenin-GOF together with a most common alteration in ccRCC, Vhl-LOF. An inducible Pax8-rtTA-LC1-Cre was used to drive recombination specifically in adult kidney epithelial cells. We confirmed mutagenesis of ß-catenin, Notch and Vhl alleles on DNA, protein and mRNA target gene levels. Surprisingly, we observed symptoms of chronic kidney disease (CKD) in mutant mice, but no increased proliferation and tumorigenesis. Thus, the responses of kidney stem cells in the tubuloid and genetic systems produced different phenotypes, i.e. enhanced renewal versus CKD.

Companion animal organoid technology to advance veterinary regenerative medicine.

First year medical and veterinary students are made very aware that drugs can have very different effects in various species or even in breeds of one specific species. On the other hand, the "One Medicine" concept implies that therapeutic and technical approaches are exchangeable between man and animals. These opposing views on the (dis)similarities between human and veterinary medicine are magnified in regenerative medicine. Regenerative medicine promises to stimulate the body's own regenerative capacity via activation of stem cells and/or the application of instructive biomaterials. Although the potential is enormous, so are the hurdles that need to be overcome before large scale clinical implementation is realistic. It is in the advancement of regenerative medicine that veterinary regenerative medicine can play an instrumental and crucial role. This review describes the discovery of (adult) stem cells in domesticated animals, mainly cats and dogs. The promise of cell-mediated regenerative veterinary medicine is compared to the actual achievements, and this will lead to a set of unanswered questions (controversies, research gaps, potential developments in relation to fundamental, pre-clinical, and clinical research). For veterinary regenerative medicine to have impact, either for human medicine and/or for domesticated animals, answering these questions is pivotal.

Prediction of outcome after ligation or thin film banding of extrahepatic shunts, based on plasma albumin concentration and hematologic expression of 8 target genes in 85 dogs.

BACKGROUND: In dogs with a congenital extrahepatic portosystemic shunt (EHPSS), outcome after surgical attenuation is difficult to predict. OBJECTIVES: Develop a minimally invasive test to predict outcome after surgical EHPSS attenuation and establish risk factors for postattenuation seizures (PAS). ANIMALS: Eighty-five client-owned dogs referred for surgical attenuation of a single EHPSS. METHODS: mRNA expression of 8 genes was measured in preoperatively collected venous blood samples. Outcome was determined at a median of 92 days (range, 26-208) postoperatively by evaluating clinical performance, blood test results and abdominal ultrasonography. Multivariable logistic regression was used to construct models predicting clinical and complete recovery. The associations between putative predictors and PAS were studied using univariable analyses. RESULTS: Five of 85 dogs developed PAS. Risk factors were age, white blood cell (WBC) count and expression of hepatocyte growth factor activator and LysM and putative peptidoglycan-binding domain-containing protein 2. Clinical recovery was observed in 72 of 85 dogs and complete recovery in 51 of 80 dogs (median follow-up, 92 days). The model predicting clinical recovery included albumin, WBC count, and methionine adenosyltransferase 2 alpha (MAT2α) expression, whereas the model predicting complete recovery included albumin, and connective tissue growth factor precursor and MAT2α expression. The areas under the receiver operating characteristic curves were 0.886 (95% confidence interval [CI]: 0.783, 0.990) and 0.794 (95% CI: 0.686, 0.902), respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Two models were constructed for predicting outcome after EHPSS attenuation using venous blood samples. The model predicting clinical recovery showed the best diagnostic properties. Clinical application requires further validation.

A Century of Progress on Wilson Disease and the Enduring Challenges of Genetics, Diagnosis, and Treatment.

Wilson disease (WD) is a rare, inherited metabolic disorder manifested with varying clinical presentations including hepatic, neurological, psychiatric, and ophthalmological features, often in combination. Causative mutations in the ATP7B gene result in copper accumulation in hepatocytes and/or neurons, but clinical diagnosis remains challenging. Diagnosis is complicated by mild, non-specific presentations, mutations exerting no clear effect on protein function, and inconclusive laboratory tests, particularly regarding serum ceruloplasmin levels. As early diagnosis and effective treatment are crucial to prevent progressive damage, we report here on the establishment of a global collaboration of researchers, clinicians, and patient advocacy groups to identify and address the outstanding challenges posed by WD.

Fibroblast growth factor-21 (FGF21) analogs as possible treatment options for diabetes mellitus in veterinary patients.

Fibroblast growth factors (FGFs) are involved in numerous metabolic processes. The endocrine subfamily of FGFs, consisting of FGF19, FGF21, and FGF23, might have beneficial effects in the treatment of diabetes mellitus (DM) and/or obesity. The analog with the greatest potential, FGF21, lowers blood glucose levels, improves insulin sensitivity, and induces weight loss in several animal models. In this review we summarize recent (pre)clinical findings with FGF21 analogs in animal models and men. Furthermore, possible applications of FGF21 analogs for pets with DM will be discussed. As currently, information about the use of FGF21 analogs in pet animals is scarce.

Human Cholangiocytes Form a Polarized and Functional Bile Duct on Hollow Fiber Membranes.

Liver diseases affect hundreds of millions of people worldwide; most often the hepatocytes or cholangiocytes are damaged. Diseases of the biliary tract cause severe patient burden, and cholangiocytes, the cells lining the biliary tract, are sensitive to numerous drugs. Therefore, investigations into proper cholangiocyte functions are of utmost importance, which is restricted, in vitro, by the lack of primary human cholangiocytes allowing such screening. To investigate biliary function, including transepithelial transport, cholangiocytes must be cultured as three-dimensional (3D) ductular structures. We previously established murine intrahepatic cholangiocyte organoid-derived cholangiocyte-like cells (CLCs) and cultured them onto polyethersulfone hollow fiber membranes (HFMs) to generate 3D duct structures that resemble native bile ducts at the structural and functional level. Here, we established an efficient, stepwise method for directed differentiation of human intrahepatic cholangiocyte organoids (ICOs) into CLCs. Human ICO-derived CLCs showed key characteristics of cholangiocytes, such as the expression of structural and functional markers, formation of primary cilia, and P-glycoprotein-mediated transport in a polarized fashion. The organoid cultures exhibit farnesoid X receptor (FXR)-dependent functions that are vital to liver bile acid homeostasis in vivo. Furthermore, human ICO-derived CLCs cultured on HFMs in a differentiation medium form tubular architecture with some tight, confluent, and polarized monolayers that better mimic native bile duct characteristics than differentiated cultures in standard 2D or Matrigel-based 3D culture plates. Together, our optimized differentiation protocol to obtain CLC organoids, when applied on HFMs to form bioengineered bile ducts, will facilitate studying cholangiopathies and allow developing therapeutic strategies.

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.

COMMD1, a multi-potent intracellular protein involved in copper homeostasis, protein trafficking, inflammation, and cancer.

Copper is a trace element indispensable for life, but at the same time it is implicated in reactive oxygen species formation. Several inherited copper storage diseases are described of which Wilson disease (copper overload, mutations in ATP7B gene) and Menkes disease (copper deficiency, mutations in ATP7A gene) are the most prominent ones. After the discovery in 2002 of a novel gene product (i.e. COMMD1) involved in hepatic copper handling in Bedlington terriers, studies on the mechanism of action of COMMD1 revealed numerous non-copper related functions. Effects on hepatic copper handling are likely mediated via interactions with ATP7B. In addition, COMMD1 has many more interacting partners which guide their routing to either the plasma membrane or, often in an ubiquitination-dependent fashion, trigger their proteolysis via the S26 proteasome. By stimulating NF-κB ubiquitination, COMMD1 dampens an inflammatory reaction. Finally, targeting COMMD1 function can be a novel approach in the treatment of tumors.

COMMD1 Exemplifies the Power of Inbred Dogs to Dissect Genetic Causes of Rare Copper-Related Disorders.

Wilson's Disease is a rare autosomal recessive disorder in humans, often presenting with hepatic copper overload. Finding the genetic cause of a rare disease, especially if it is related to food constituents like the trace element copper, is a Herculean task. This review describes examples of how the unique population structure of in-bred dog strains led to the discovery of a novel gene and two modifier genes involved in inherited copper toxicosis. COMMD1, after the discovery in 2002, was shown to be a highly promiscuous protein involved in copper transport, protein trafficking/degradation, regulation of virus replication, and inflammation. Mutations in the ATP7A and ATP7B proteins in Labrador retrievers and Dobermann dogs resulted in a wide variation in hepatic copper levels in these breeds. To our knowledge, numerous dog breeds with inherited copper toxicosis of unknown genetic origin exist. Therefore, the possibility that men's best friend will provide new leads in rare copper storage diseases seems realistic.

Hippo signaling pathway in companion animal diseases, an under investigated signaling cascade.

The Hippo pathway is a highly conserved kinase cascade in mammals with the proteins YAP and TAZ as its most important downstream effectors that shuttle between cytoplasma and nucleus. It has a crucial role in processes such as embryogenesis, organ size control, homeostasis and tissue regeneration, where mechanosensing and/or cell-cell interactions are involved. As the pathway is associated with many essential functions in the body, its dysregulation is related to many diseases. In contrast to human pathology, a PubMed-search on Hippo, YAP/TAZ and companion animals (horse, equine, dog, canine, cat, feline) retrieved few publications. Because of its high level of functional conservation, it is anticipated that also in veterinary sciences aberrant Hippo YAP/TAZ signaling would be implicated in animal pathologies. Publications on Hippo YAP/TAZ in companion animals are mainly in cats and dogs and related to oncology. Here, we emphasize the important role of YAP/TAZ in liver diseases. First the liver has a remarkable regeneration capacity and a strict size control and the liver has a moderate liver cell renewal (homeostasis). The last years numerous papers show the importance of YAP/TAZ in hepatocellular carcinoma (HCC), hepatocyte differentiation and bile duct epithelial (BEC) cell survival. YAP/TAZ signaling is involved in activation of hepatic stellate cells crucial in fibrogenesis. The availability of drugs (e.g. verteporfin) targeting the YAP/TAZ pathway are described as is their potential usage in veterinary medicine. The aim of this overview is to stimulate researchers' and clinicians' interest in the potential role of Hippo YAP/TAZ signaling in veterinary medicine.

Tissue-Engineered Bile Ducts for Disease Modeling and Therapy.

Recent biotechnical advances in the in vitro culture of cholangiocytes and generation of bioengineered biliary tissue have a high potential for creating biliary tissue to be used for disease modeling, drug screening, and transplantation. For the past few decades, scientists have searched for a source of cholangiocytes, focused on primary cholangiocytes or cholangiocytes derived from hepatocytes or stem cells. At the same time, the development of scaffolds for biliary tissue engineering for transplantation and modeling of cholangiopathies has been explored. In this review, we provide an overview on the current understanding of cholangiocytes sources, the effect of signaling molecules, and transcription factors on cell differentiation, along with the effects of extracellular matrix molecules and scaffolds on bioengineered biliary tissues, and their application in disease modeling and drug screening. Impact statement Over the past few decades, biliary tissue engineering has acquired significant attention, but currently a number of factors hinder this field to eventually generate bioengineered bile ducts that mimic in vivo physiology and are suitable for transplantation. In this review, we present the latest advances with respect to cell source selection, influence of growth factors and scaffolds, and functional characterization, as well as applications in cholangiopathy modeling and drug screening. This review is suited for a broad spectrum of readers, including fundamental liver researchers and clinicians with interest in the current state and application of bile duct engineering and disease modeling.

MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments.

The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles.